Systems and methods for biomolecule retention

ABSTRACT

Compositions, systems, and methods for the display of analytes such as biomolecules are described. Display of analytes is achieved by coupling of the analytes to displaying molecules that are configured to associate with surfaces or interfaces. Arrays of analytes may be formed from the described systems for utilization in assays and other methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/692,035, filed Mar. 10, 2022, which claims priority to U.S.Provisional Application No. 63/159,500, filed on Mar. 11, 2021, and U.S.Provisional Application No. 63/256,761, filed on Oct. 18, 2021, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in XML format and is hereby incorporated by reference inits entirety. Said XML copy, created on Oct. 27, 2022, is named50109_4005US02.xml and is 574,343 bytes in size.

BACKGROUND OF THE INVENTION

Analytes and other molecules may be formed into structured or orderedarrays for various purposes, including for analytical techniques andother chemical purposes. For example, biomolecules may be patterned intosingle-molecule arrays for purposes such as sequencing or moleculeidentification. High efficiency of analyte deposition on single-moleculearrays may benefit from methods of preparing analytes and preparingsurfaces or interfaces where the analytes are to be deposited.

SUMMARY OF THE INVENTION

In an aspect, provided herein is a composition, comprising: a structurednucleic acid particle (SNAP) comprising (i) a display moiety that isconfigured to couple to an analyte, (ii) a capture moiety that isconfigured to couple with a surface, and (iii) a multifunctional moietycomprising a first functional group and a second functional group,wherein the multifunctional moiety is coupled to the structured nucleicacid particle, and wherein the first functional group is coupled to thedisplay moiety, and wherein the second functional group is coupled tothe capture moiety.

In another aspect, provided herein is a composition, comprising: astructured nucleic acid particle, and a multifunctional moiety, whereinthe multifunctional moiety is coupled to the SNAP, and wherein themultifunctional moiety is configured to form a continuous linker from asurface to an analyte.

In another aspect, provided herein is a structured nucleic acid particle(SNAP) complex, comprising two or more SNAPs, wherein each SNAP of thetwo or more SNAPs is selected independently from the group consisting ofa display SNAP, a utility SNAP, or a combination thereof, wherein thedisplay SNAP comprises a display moiety that is configured to couple toan analyte, wherein the utility SNAP comprises a capture moiety that isconfigured to couple with a surface, and wherein the two or more SNAPsare coupled to form the SNAP complex.

In another aspect, provided herein is a structured nucleic acid particle(SNAP) composition, comprising: a material comprising a surface, and twoor more SNAPs, wherein each SNAP of the two or more SNAPs is selectedindependently from the group consisting of a display SNAP, a utilitySNAP, or a combination thereof, wherein the display SNAP comprises adisplay moiety that is configured to couple to an analyte, wherein thetwo or more SNAPs are coupled to the surface, and wherein a first SNAPof the two or more SNAPs is coupled to a second SNAP of the two or moreSNAPs, thereby forming a SNAP complex.

In another aspect, provided herein is a composition, comprising: a) ananalyte, b) a display SNAP, and c) one or more SNAPs selected from thegroup consisting of a display SNAP, a utility SNAP, and combinationsthereof, wherein the display SNAP comprises a display moiety that isconfigured to couple to the analyte, wherein the display SNAP is coupledto the analyte, and wherein the display SNAP is coupled to the one ormore SNAPs, thereby forming a SNAP complex.

In another aspect, provided herein is a structured nucleic acid particlecomposition, comprising: a) a material comprising a surface, b) ananalyte, c) a display SNAP, and one or more SNAPs selected from thegroup consisting of a display SNAP, a utility SNAP, and combinationsthereof, wherein the display SNAP comprises a display moiety that isconfigured to couple to the analyte, wherein the display SNAP is coupledto the analyte, wherein the display SNAP is coupled to the one or moreSNAPs, thereby forming a SNAP complex, and wherein the SNAP complex iscoupled to the surface.

In another aspect, provided herein is an array, comprising: a) aplurality of SNAP complexes, and b) a material comprising a surface,wherein each of the SNAP complexes is coupled to the surface, whereineach SNAP complex of the plurality of SNAP complexes is coupled to oneor more other SNAP complexes of the plurality of SNAP complexes, andwherein each SNAP complex of the plurality of SNAP complexes comprisestwo or more SNAPs selected independently from the group consisting of adisplay SNAP, a utility SNAP, and combinations thereof.

In another aspect, provided herein is a method of forming an array,comprising: a) providing a plurality of SNAP complexes, b) coupling eachSNAP complex of the plurality of SNAP complexes to one or moreadditional SNAP complexes from the plurality of SNAP complexes, and c)coupling each SNAP complex of the plurality of SNAP complexes with asurface, wherein each SNAP complex comprises a display SNAP and one ormore utility SNAPs, and wherein each SNAP complex comprises a couplingmoiety that couples with the surface, thereby forming an array.

In another aspect, provided herein is a composition, comprising: a) astructured nucleic acid particle, wherein the structured nucleic acidparticle comprises: i) a retaining component; ii) a display moietycomprising a coupling group that is configured to couple an analyte,wherein the display moiety is coupled to the retaining component, andiii) a capture moiety that is configured to couple with a surface,wherein the capture moiety comprises a plurality of firstsurface-interacting oligonucleotides, and wherein each firstsurface-interacting oligonucleotide of the plurality of firstsurface-interacting oligonucleotides comprises a first nucleic acidstrand that is coupled to the retaining component and a firstsurface-interacting moiety, wherein the first surface-interacting moietyis configured to form a coupling interaction with a surface-linkedmoiety, wherein the capture moiety is restrained from contacting thedisplay moiety by the retaining component, and b) an analyte comprisinga complementary coupling group that is configured to couple to thedisplay moiety of the structured nucleic acid particle.

In another aspect, provided herein is a composition, comprising: a) astructured nucleic acid particle, wherein the structured nucleic acidparticle comprises: i) a retaining component; ii) a display moiety thatis coupled to the retaining component; and iii) a capture moiety that iscoupled to the retaining component, wherein the capture moiety comprisesa plurality of oligonucleotides, and wherein each oligonucleotide of theplurality of oligonucleotides comprises a surface-interacting moiety,and b) a solid support comprising a coupling surface, wherein thesurface comprises a surface-linked moiety, and wherein asurface-interacting moiety of the plurality of surface-interactingmoieties is coupled to the surface-linked, wherein the display moiety isrestrained from contacting the surface by the retaining component.

In another aspect, provided herein is a method of identifying apolypeptide, the method comprising: a) providing a SNAP composition asset forth herein, wherein the polypeptide is coupled to the displaymoiety, b) contacting the solid support with a plurality of detectableaffinity reagents, c) detecting presence or absence of binding of thedetectable affinity reagent of the plurality of detectable affinityagents to the polypeptide, d) optionally repeating steps b)-c) with asecond plurality of detectable affinity reagents, and e) based upon thepresence or absences of binding of one or more of the affinity reagents,identifying the polypeptide.

In another aspect, provided herein is a method of sequencing apolypeptide, the method comprising: a) providing a SNAP composition asset forth herein, wherein the polypeptide is coupled to the displaymoiety, b) removing a terminal amino acid residue of the polypeptide byan Edman-type degradation reaction, c) identifying the terminal aminoacid residue, and d) repeating steps b-c) until a sequence of amino acidresidues has been identified for the polypeptide.

In another aspect, provided herein is a single-analyte array,comprising: a) a solid support comprising a plurality of addresses,wherein each address of the plurality of addresses is resolvable atsingle-analyte resolution, wherein each address comprises a couplingsurface, and wherein each coupling surface comprises one or moresurface-linked moieties, b0 a plurality of structured nucleic acidparticles, wherein each structured nucleic acid particle comprises acoupling moiety, wherein the coupling moiety comprises a plurality ofoligonucleotides, wherein each oligonucleotide of the plurality ofoligonucleotides comprises a surface-interacting moiety, wherein eachstructured nucleic acid particle of the plurality of structured nucleicacid particles is coupled to an address of the plurality of addresses bya binding of the surface-interacting moiety of the plurality ofoligonucleotides to a surface-linked moiety of the one or morecomplementary oligonucleotides, and wherein a structured nucleic acidparticle of the plurality of structured nucleic acid particles comprisesa display moiety comprising a coupling site that is coupled to ananalyte.

In another aspect, provided herein is a single-analyte array,comprising: a) a solid support comprising a plurality of addresses,wherein each address of the plurality of addresses is resolvable fromeach other address at single-analyte resolution, and wherein eachaddress is separated from each adjacent address by one or moreinterstitial regions, and b) a plurality of analytes, wherein a singleanalyte of the plurality of analytes is coupled to an address of theplurality of addresses, wherein each address of the plurality ofaddresses comprises no more than one single analyte, wherein each singleanalyte is coupled to a coupling surface of the address by a nucleicacid structure, and wherein the nucleic acid structure occludes thesingle analyte from contacting the coupling surface.

In another aspect, provided herein is a nucleic acid nanostructure,comprising at least 10 coupled nucleic acids, wherein the nucleic acidnanostructure comprises: a) a compacted region comprising a highinternal complementarity, wherein the high internal complementaritycomprises at least 50% double-stranded nucleic acids and at least 1%single-stranded nucleic acids, and wherein the compacted regioncomprises a display moiety, wherein the display moiety is coupled to, orconfigured to couple to, an analyte of interest; and b) a perviousregion comprising a low internal complementarity, wherein the lowinternal complementarity comprises at least about 50% single-strandednucleic acids, and wherein the pervious region comprises a couplingmoiety, wherein the coupling moiety forms, or is configured to form, acoupling interaction with a solid support.

In another aspect, provided herein is a nucleic acid nanostructure,comprising: a) a compacted structure, wherein the compacted structurecomprises a scaffold strand and a first plurality of stapleoligonucleotides, wherein at least 80% of nucleotides of the scaffoldstrand are hybridized to nucleotides of the first plurality of stapleoligonucleotides, wherein the first plurality of staple oligonucleotideshybridizes to the scaffold strand to form a plurality of tertiarystructures, wherein the plurality of tertiary structures includesadjacent tertiary structures linked by a single-stranded nucleic acidregion of the scaffold, and wherein a relative position of an adjacenttertiary structure of the adjacent tertiary structures is positionallyconstrained; and b) a pervious structure, wherein the pervious structurecomprises a second plurality of staple oligonucleotides, wherein thestaple oligonucleotides are coupled to the scaffold strand of thecompacted structure, wherein the pervious structure comprises at least50% single-stranded nucleic acid, and wherein the pervious structure hasan anisotropic three-dimensional distribution around at least a portionof the compacted structure.

In another aspect, provided herein is a nucleic acid nanostructure,comprising: a) a compacted structure, wherein the compacted structurecomprises a scaffold strand and a first plurality of stapleoligonucleotides, wherein at least 80% of nucleotides of the scaffoldstrand are hybridized to nucleotides of the first plurality of stapleoligonucleotides, wherein the first plurality of staple oligonucleotideshybridizes to the scaffold strand to form a plurality of tertiarystructures, wherein the plurality of tertiary structures includesadjacent tertiary structures linked by a single-stranded region of thescaffold strand, wherein the relative positions of the adjacent tertiarystructures are positionally constrained, and wherein the compactedstructure comprises an effective surface area; and b) a perviousstructure, wherein the pervious structure comprises a second pluralityof staple oligonucleotides, wherein the staple oligonucleotides arecoupled to the scaffold strand of the compacted structure, and whereinthe pervious structure comprises at least 50% single-stranded nucleicacid; and wherein (i) an effective surface area of the nucleic acidnanostructure is larger than the effective surface area of the compactedstructure, or ii) the ratio of effective surface area to volume of thenucleic acid nanostructure is larger than the ratio of effective surfacearea to volume of the compacted structure.

In another aspect, provided herein is a nucleic acid nanostructure,comprising a plurality of nucleic acid strands, wherein each nucleicacid strand of the plurality of nucleic acid strands is hybridized toanother nucleic acid strand of the plurality of nucleic acid strands toform a plurality of tertiary structures, and wherein a nucleic acidstrand of the plurality of nucleic acid strands comprises a firstnucleotide sequence that is hybridized to a second nucleic acid strandof the plurality of nucleic acid strands, wherein the nucleic acidstrand of the plurality of nucleic acid strands further comprises asecond nucleotide sequence of at least 100 consecutive nucleotides, andwherein at least 50 nucleotides of the second nucleotide sequence issingle-stranded.

In another aspect, provided herein is a composition, comprising: a) asolid support comprising a plurality of sites; and b) a plurality ofstructured nucleic acid particles (SNAPs), in which each SNAP is coupledto, or is configured to couple to, an analyte, and in which each SNAP ofthe plurality of SNAPs is coupled to a site of the plurality of sites,wherein the plurality of sites comprises a first subset comprising afirst quantity of sites and a second subset comprising a second quantityof sites, in which each site of the first subset comprises two or morecoupled SNAPs, in which each site of the second subset comprises one andonly one coupled SNAP, and in which a ratio of the quantity of sites ofthe first subset to the quantity of sites of the second subset is lessthan a ratio predicted by a Poisson distribution.

In another aspect, provided herein is an analyte array, comprising: a) asolid support comprising a plurality of sites; and b) a plurality ofnucleic acid nanostructures, wherein each nucleic acid nanostructure iscoupled to an analyte of interest, and wherein each nucleic acidnanostructure of the plurality of nucleic acid nanostructures is coupledto a site of the plurality of sites, wherein at least 40% of sites ofthe plurality of sites comprise one and only one analyte of interest.

In another aspect, provided herein is a composition comprising: a) asolid support comprising a site that is configured to couple a nucleicacid nanostructure; and b) the nucleic acid nanostructure, wherein thenucleic acid nanostructure is coupled to the site, wherein the nucleicacid nanostructure is coupled to an analyte of interest; and wherein thenucleic acid nanostructure is configured to prevent contact between theanalyte of interest and the solid support.

In another aspect, provided herein is a composition, comprising: a) asolid support comprising a site that is configured to couple a nucleicacid nanostructure, wherein the site comprises a surface area; and b)the nucleic acid nanostructure, wherein the nucleic acid nanostructureis coupled to the site, wherein the nucleic acid nanostructure iscoupled to, or is configured to couple to, an analyte of interest;wherein the nucleic acid nanostructure comprises a total effectivesurface area in an unbound configuration, wherein the nucleic acidnanostructure comprises a compact structure with an effective surfacearea, wherein the effective surface area of the compacted structure inthe unbound configuration is less than 50% of the surface area of thesite, and wherein the unbound configuration comprises the nucleic acidnanostructure being uncoupled from the site.

In another aspect, provided herein is a method of coupling a nucleicacid nanostructure to an array site, comprising: a) contacting an arraycomprising a site with a nucleic acid nanostructure, wherein the sitecomprises a plurality of surface-linked moieties, and wherein thenucleic acid nanostructure comprises a plurality of capture moieties; b)coupling the nucleic acid nanostructure to the site in an initialconfiguration, wherein the initial configuration does not comprise astable configuration, and wherein the nucleic acid nanostructure iscoupled by a coupling of a capture moiety of the plurality of capturemoieties to a surface-linked moiety of the plurality of surface-linkedmoieties; c) uncoupling the coupling of the capture moiety of theplurality of capture moieties to the surface-linked moiety of theplurality of surface-linked moieties; and d) altering the nucleic acidnanostructure from the initial configuration to the stableconfiguration, wherein each capture moiety of the plurality of capturemoieties is coupled to a surface-linked moiety of the plurality ofsurface-linked moieties.

In another aspect provided herein is a method of forming a multiplexarray of analytes, comprising: a) contacting an array comprising aplurality of sites with a first plurality of nucleic acidnanostructures, wherein each nucleic acid nanostructure of the firstplurality of nucleic acid nanostructures is coupled to an analyte ofinterest of a first plurality of analytes of interest; b) contacting thearray comprising the plurality of sites with a second plurality ofnucleic acid nanostructures, wherein each nucleic acid nanostructure ofthe second plurality of nucleic acid nanostructures is coupled to ananalyte of interest of a second plurality of analytes of interest; c)depositing the first plurality of nucleic acid nanostructures at a firstsubset of sites of the plurality of sites; and d) depositing the secondplurality of nucleic acid nanostructures at a second subset of sites ofthe plurality of sites, wherein the first subset of sites and the secondsubset of sites comprise a random spatial distribution.

In another aspect, provided herein is a nanostructure, comprising: a) acompacted nucleic acid structure comprising a scaffold strand hybridizedto a first plurality of staple oligonucleotides, wherein the firstplurality of staple oligonucleotides hybridizes to the scaffold strandto form a plurality of tertiary structures, wherein the plurality oftertiary structures comprises adjacent tertiary structures linked by asingle-stranded region of the scaffold strand, and wherein relativepositions of the adjacent tertiary structures are positionallyconstrained; b) a pervious structure, wherein the pervious structurecomprises a second plurality of staple oligonucleotides hybridized tothe scaffold strand; and c) a solid support comprising surface-linkedoligonucleotides, wherein the surface-linked oligonucleotides areattached to a surface of the solid support, and wherein thesurface-linked oligonucleotides are hybridized to stapleoligonucleotides of the pervious structure.

In another aspect, provided herein is a method of coupling a nucleicacid nanostructure to an array, comprising: a) contacting a solidsupport with a nucleic acid nanostructure, wherein the solid supportcomprises surface-linked oligonucleotides attached to the solid support,and wherein the nucleic acid nanostructure comprises: i) a compactednucleic acid structure comprising a scaffold strand hybridized to afirst plurality of staple oligonucleotides, wherein the first pluralityof staple oligonucleotides hybridizes to the scaffold strand to form aplurality of tertiary structures, wherein the plurality of tertiarystructures comprises adjacent tertiary structures linked by asingle-stranded region of the scaffold strand, and wherein relativepositions of the adjacent tertiary structures are positionallyconstrained; and ii) a pervious structure, wherein the perviousstructure comprises a second plurality of staple oligonucleotideshybridized to the scaffold strand; and b) hybridizing a surface-linkedoligonucleotide to a staple oligonucleotide of the second plurality ofstaple oligonucleotides.

In another aspect, provided herein is a method of preparing an array ofanalytes, comprising: a) providing an array comprising a plurality ofsites, wherein each site comprises surface-linked oligonucleotides; b)contacting the array with a plurality of analytes, wherein each analyteis coupled to a nucleic acid nanostructure, wherein each nucleic acidnanostructure comprises a plurality of surface-couplingoligonucleotides; and c) coupling one and only one nucleic acidnanostructure to a site of the plurality of sites, wherein coupling thenucleic acid nanostructure comprises hybridizing a surface-linkedoligonucleotide of the site to the surface-coupling oligonucleotide ofthe nucleic acid nanostructure.

In another aspect, provided herein is an array of analytes of interest,comprising: a) a solid support comprising a plurality of sites, whereineach site comprises surface-linked oligonucleotides; b) a plurality ofnucleic acid nanostructures, wherein each nucleic acid nanostructure isconfigured to couple an analyte, wherein each nucleic acid nanostructurecomprises a plurality of surface-coupling oligonucleotides, wherein eachsurface-coupling oligonucleotide comprises no self-complementarity, andwherein each nucleic acid nanostructure of the plurality of nucleic acidnanostructures is coupled to a site of the plurality of sites by ahybridizing of a surface-coupling oligonucleotide to a surface-linkedoligonucleotide; and c) a plurality of analytes of interest, in whicheach analyte of interest is coupled to a nucleic acid nanostructure ofthe plurality of nucleic acid nanostructures.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Novel features of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the present invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1A illustrates angular offset for two faces of a structured nucleicacid particle (SNAP), in accordance with some embodiments. FIG. 1Billustrates angular offset for two faces of a SNAP, in accordance withsome embodiments.

FIG. 2A depicts two sets of tertiary structures in a SNAP, in accordancewith some embodiments. FIG. 2B shows a cross-section of a SNAP withmultiple faces, in accordance with some embodiments. FIG. 2C depicts twosets of tertiary structures in a SNAP, in accordance with someembodiments. FIG. 2D shows a cross-section of a SNAP with multiplefaces, in accordance with some embodiments.

FIG. 3A shows a SNAP comprising a multifunctional moiety, in accordancewith some embodiments. FIG. 3B shows a linking moiety of amultifunctional moiety, in accordance with some embodiments. FIG. 3Cshows a SNAP comprising a multifunctional moiety coupled to a solidsupport, in accordance with some embodiments. FIG. 3D shows an analytecoupled to a solid support by a multifunctional moiety, in accordancewith some embodiments.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H show a SNAP coupled to asurface, in accordance with some embodiments.

FIGS. 5A, 5B, 5C, and 5D illustrate a SNAP with different capture faceconformations, in accordance with some embodiments.

FIG. 6 depicts a square-shaped SNAP, in accordance with someembodiments.

FIG. 7A shows a multifunctional moiety comprising an alkyl group, inaccordance with some embodiments. FIG. 7B shows a multifunctional moietycomprising modified oligonucleotides, in accordance with someembodiments.

FIGS. 8A, 8B, 8C, and 8D illustrate a SNAP comprising a multifunctionalmoiety, in accordance with some embodiments.

FIGS. 9A, 9B, 9C, 9D, 9E, and 9F illustrate a method of coupling ananalyte to a surface, in accordance with some embodiments.

FIGS. 10A, 10B, 10C, and 10D depict a SNAP comprising twomultifunctional moieties, in accordance with some embodiments.

FIGS. 11A, 11B, 11C, and 11D illustrate a SNAP comprising amultifunctional moiety, in accordance with some embodiments.

FIGS. 12A, 12B, and 12C show a SNAP complex comprising tile-shapedSNAPs, in accordance with some embodiments.

FIGS. 13A, 13B, 13C, and 13D depict differing SNAP symmetries, inaccordance with some embodiments.

FIGS. 14A and 14B illustrate a three-dimensional SNAP conformation, inaccordance with some embodiments.

FIGS. 15A and 15B show different orientation of coupled SNAPs, inaccordance with some embodiments.

FIGS. 16A and 16B depict a three-dimensional SNAP complex, in accordancewith some embodiments.

FIGS. 17A, 17B, and 17C show an array formed from SNAP complexes, inaccordance with some embodiments.

FIGS. 18A, 18B, and 18C show an array formed from SNAP complexes, inaccordance with some embodiments.

FIGS. 19A and 19B depict a complex of SNAPs formed at an interface, inaccordance with some embodiments.

FIG. 20 depicts a method of isolating analyte fractions onto differentSNAP species, in accordance with some embodiments.

FIGS. 21A and 21B show SNAP-protein conjugate deposition on a patternedarray.

FIG. 22 illustrates an array comprising multiple species of SNAPs, inaccordance with some embodiments.

FIGS. 23A and 23B illustrate an array comprising multiple species ofSNAPs, in accordance with some embodiments.

FIG. 24 illustrates an array comprising multiple species of SNAPs, inaccordance with some embodiments.

FIGS. 25A, 25B, and 25C depict a SNAP complex on a surface comprisingsurface roughness, in accordance with some embodiments.

FIGS. 26A, 26B, and 26C depict multiple SNAP complexes on a singlebinding site, in accordance with some embodiments.

FIGS. 27A and 27B show an array containing patterned binding sites, inaccordance with some embodiments.

FIGS. 28A and 28B illustrate a SNAP complex coupling to a patternedsurface, in accordance with some embodiments.

FIG. 29 depicts a three-dimensional SNAP complex, in accordance withsome embodiments.

FIGS. 30A, 30B, 30C, and 30D show HPLC data for SNAP-protein conjugatepurification.

FIG. 31 gives a schematic view of a 5-tile DNA origami SNAP, inaccordance with some embodiments.

FIGS. 32A, 32B, 32C, 32D, 32E, and 32F show fluorescent confocalscanning microscopy image data for SNAP deposition.

FIG. 33 plots SNAP deposition under differing solvent conditions.

FIGS. 34A, 34B, and 34C show fluorescent confocal scanning microscopyimage data for SNAP deposition.

FIG. 35 plots SNAP deposition under differing solvent conditions.

FIGS. 36A and 36B illustrate a scheme for producing SNAPs, in accordancewith some embodiments.

FIGS. 37A and 37B depict a SNAP comprising regions of full structuringand partial structuring, in accordance with some embodiments.

FIGS. 38A and 38B depict a SNAP comprising a multivalent moiety in aninternal volume region, in accordance with some embodiments.

FIGS. 39A and 39B depict a SNAP comprising a chemically-modifiedinternal volume region, in accordance with some embodiments.

FIGS. 40A, 40B, and 40C illustrate various configurations of a SNAPcontaining a plurality of surface-interacting moieties in contact with acoupling surface comprising a plurality of surface-linked moieties, inaccordance with some embodiments.

FIGS. 41A and 41B shows differing distributions of surface-interactingmoieties on a capture moiety of a SNAP, in accordance with someembodiments.

FIG. 42 depicts a scheme for providing a plurality of surface-linkedmoieties to a solid support for the purpose of facilitating bindinginteractions with a SNAP, in accordance with some embodiments.

FIG. 43 shows detection of His-12 peptide SNAP arrays by B1 aptamerprobes for double His-12 SNAPs on oligonucleotide-coated surfaces.

FIG. 44 shows detection of His-12 peptide SNAP arrays by B1 aptamerprobes for single His-12 SNAPs on oligonucleotide-coated surfaces.

FIG. 45 shows a comparison of His-12 detection by B1 aptamer probes forSNAPs on APTMS-coated and oligonucleotide-containing surfaces.

FIG. 46 displays fluorescent imaging data for unpatterned SNAP arraysformed on glass surfaces containing different surface concentrations ofoligonucleotides and differing SNAP concentrations.

FIG. 47 displays fluorescent imaging data for unpatterned SNAPs arraysformed by direct conjugation of SNAPs to azide-containing surfaces.

FIG. 48 depicts a difference between an effective surface area and afootprint of a nucleic acid, in accordance with some embodiments.

FIGS. 49A, 49B, 49C, 49D, and 49E illustrate aspects of nucleic acidstructure and conformation, in accordance with some embodiments.

FIGS. 50A, 50B, 50C, 50D, 50E, and 50F show steps of a method forforming a multiplexed single-analyte array, in accordance with someembodiments.

FIG. 51 displays a nucleic acid nanostructure comprising a scaffoldstrand and a plurality of staple oligonucleotides, in accordance withsome embodiments.

FIGS. 52A, 52B, 52C, 52D, 52E, 52F, 52G, and 52H depict variousconfigurations of nucleic acid nanostructures comprising compactedstructures and pervious structures, in accordance with some embodiments.

FIGS. 53A, 53B, 53C, 53D, and 53E illustrate various configurations ofnucleic acid nanostructures comprising pervious structures that areconfigured to form multi-valent binding interactions, in accordance withsome embodiments.

FIGS. 54A, 54B, and 54C show methods for forming nucleic acidnanostructures with pervious structures, in accordance with someembodiments.

FIGS. 55A, 55B, 55C, and 55D display methods for forming multi-valentbinding interactions between a nucleic acid nanostructure and a solidsupport, in accordance with some embodiments.

FIGS. 56A, 56B, and 56C depict various configurations of nucleic acidnanostructures comprising pervious structures, in which the perviousstructures form multi-valent binding interactions with a solid support,in accordance with some embodiments.

FIG. 57 illustrates a change in conformation for a nucleic acidnanostructure due to a surface-binding interaction, in accordance withsome embodiments.

FIGS. 58A, 58B, and 58C show a method of reconfiguring a bindingconfiguration of a nucleic acid nanostructure coupled to an array site,in accordance with some embodiments.

FIGS. 59A and 59B display atomic force microscopy images of nucleic acidnanostructures. FIGS. 59C and 59D plot various measurements of nucleicacid nanostructure yield and size.

FIGS. 60A, 60B, 60C, and 60D depict various configurations of arraysites comprising two or more types of coupled surface moieties, inaccordance with some embodiments.

FIGS. 61A, 61B, 61C, 61D, and 61E display steps of a method of couplinga nucleic acid nanostructure to a solid support utilizing unreactedfunctional groups, in accordance with some embodiments.

FIGS. 62A, 62B, and 62C illustrate methods of forming arrays that areconfigured to produce multiplexed arrays of analytes, in accordance withsome embodiments. FIGS. 62D and 62E illustrate a method of depositingtwo or more types of analytes to form a multiplexed array, in accordancewith some embodiments.

FIG. 63 shows a plurality of sites of an array comprising variousdefects or disruptions, in accordance with some embodiments.

FIG. 64 depicts an array of analytes formed by a non-lithographicmethod, in accordance with some embodiments.

FIG. 65 shows a method of forming an array of analytes via acharge-mediated interaction, in accordance with some embodiments.

FIGS. 66A, 66B, 66C, and 66D displays various shapes and morphologies offormed array features in accordance with some embodiments.

FIG. 67A illustrates a schematic of a functionalized array site, inaccordance with some embodiments. FIG. 67B displays fluorescencemicroscopy characterization of an array formed by lithographicpatterning. FIG. 67C displays atomic force microscopy data of surfaceroughness of an array site formed by lithographic patterning. FIGS. 67Dand 67E plot data for average array site diameter and site pitch forarrays formed by lithographic patterning.

FIG. 68 displays fluorescence microscopy images for cycles of bindingand stripping fluorescently-labeled oligonucleotides from functionalnucleic acids.

FIGS. 69A, 69B, 69C, and 69D display fluorescence microscopy images fora multiplexed array during binding and stripping offluorescently-labeled oligonucleotides with functional acids ofstructured nucleic acid particles.

FIG. 70 displays fluorescence microscopy images for arrays comprisingfunctional nucleic acids of differing nucleotide sequence lengths duringbinding and stripping of fluorescently-labeled oligonucleotides.

DETAILED DESCRIPTION OF THE INVENTION

The ordering of molecules at the nanoscale is a critical problem fornumerous technologies, including analytical and bioanalytical methods,catalysis and biocatalysis, micro- and nanofluidics, and micro- andnano-electronics. Of particular interest are methods of arrangingmolecules at surfaces or interfaces where the length scales of surfacefeatures or surface irregularities often approach the length scale ofmolecules that are to be arranged at the surface or interface. Forexample, single-molecule analytical techniques are of interest fornumerous biological applications, including genomics, transcriptomics,and proteomics. The formation of single-analyte biomolecule arrays canbe limited by nanoscale and/or single-molecule effects that canalternately cause limited biomolecule deposition or excess biomoleculedeposition at binding sites on a single-analyte array. For example,defects in the nanoscale fabrication of solid surfaces can produce sitesthat have anomalous binding properties, thereby producing localizeddefects in array patterning. Likewise, thermodynamic effects (e.g.,entropy) and/or kinetic effects (e.g., slow dissociation) can causeunintended phenomena (e.g., molecule co-localization) at array sitesgiven a large enough sample of molecules. Consequently, in formingsingle-analyte arrays, methods of preparing consistent surfaces orinterfaces and carefully controlling the deposition of molecules on thesurfaces or interfaces is important.

It is preferable for many single-analyte, array-based techniques to formarrays that are substantially uniform, both in terms of having a singleanalyte be present at substantially all array sites of a single-analytearray (i.e., an array site occupancy value>0 analytes), and in terms ofhaving no more than one single-analyte at each array site of thesingle-analyte array (i.e., an array site occupancy value=1 analyte).The uniformity of a single-analyte array may increase as a Poisson-likeprobability distribution narrows around an array site occupancy value of1 analyte. Accordingly, array formation methods that facilitate such anarrowing of a probability mass function around an array site occupancyvalue of 1 analyte are preferable for the formation of single-analytearrays.

Intermediary particles offer a potential approach to controlling thedeposition of molecules on surfaces or interfaces. Particularly usefulintermediary particles have tunable characteristics that allow theintermediary particle to selectively interact with surfaces orinterfaces while displaying analytes and other molecules favorably on asurface or interface. Surfaces can be readily patterned usingnanofabrication techniques to create sites or addresses that areuniquely configured to capture particles set forth herein. As such, asurface can be patterned with an array of sites configured to capture aplurality of particles. By using a plurality of particles, in which eachparticle is attached to a different analyte, an array of differentanalytes can be formed on the surface and in a predetermined patternthat is suited to a desired analytical assay method, such as ananalytical method set forth herein. Exemplary intermediary particles arestructured nucleic acid particles (SNAPs), such as nucleic acid origami.The tunability of such particles arises from the helical nature ofnucleic acid tertiary structures. Over the course of a single helicalrevolution, a nucleic acid helix can orient a coupled ligand invirtually any direction over a full 360° of aspect. Consequently,structured nucleic acid particles can be engineered to display attachedmolecules at specific locations and orientations on the particle,permitting multiple attached molecules to be optimally separated andpositioned for best effect. Other nucleic acid nanostructures can besimilarly deployed as intermediate particles for displaying analytes ona surface.

Described herein are structured nucleic acid particles and systemsthereof that can be used to facilitate the formation of single-moleculearrays of analytes and other molecules. In particular configurations,the structured nucleic acid particles comprise several structuralfeatures that increase the specificity of coupling interactions onsurfaces or interfaces, or decrease the sensitivity of the particles todefects or irregularities on surfaces or interfaces, thereby permittingthe formation of more uniform single-molecule arrays. In particular,provided herein are systems comprising structured nucleic acid particlesand solid supports whose complementary chemistries encourage thecontrolled deposition of single-analyte arrays. Each structured nucleicacid particle may be coupled to one or multiple analytes of interest,permitting the formation of uniform arrays of analytes on a surface orinterface. For example, analytes of interest may be nucleic acids,proteins, metabolites or other targets of interest for analyticalcharacterization. In another example, the analytes can be reagents usedfor synthetic methods such as synthesis of nucleic acids, proteins,small molecules, candidate therapeutics, non-biological polymers, or thelike.

Also described herein are complexes that may be formed by the couplingof multiple structured nucleic acid particles. The complexes mayincrease the efficiency and control of analyte or molecule display at asurface or interface by increasing binding interactions with surfacebinding sites and/or reducing the likelihood of unwanted analyte ormolecule co-deposition at a single location on a surface or array. Insome configurations, structured nucleic acid complexes may be configuredto form a self-assembling or self-patterning arrays for the display oranalytes or other molecules.

Definitions

As used herein, the terms “nucleic acid nanostructure” or “nucleic acidnanoparticle,” refer synonymously to a single- or multi-chainpolynucleotide molecule comprising a compacted three-dimensionalstructure. The compacted three-dimensional structure can optionally havea characteristic tertiary structure. An exemplary nucleic acidnanostructure is a structured nucleic acid particle (SNAP). A SNAP canbe configured to have an increased number of interactions betweenregions of a polynucleotide strand, less distance between the regions,increased number of bends in the strand, and/or more acute bends in thestrand, as compared to the same nucleic acid molecule in a random coilor other non-structured state. Alternatively or additionally, thecompacted three-dimensional structure of a nucleic acid nanostructurecan optionally have a characteristic quaternary structure. For example,a nucleic acid nanostructure can be configured to have an increasednumber of interactions between polynucleotide strands or less distancebetween the strands, as compared to the same nucleic acid molecule in arandom coil or other non-structured state. In some configurations, thetertiary structure (i.e. the helical twist or direction of thepolynucleotide strand) of a nucleic acid nanostructure can be configuredto be more dense than the same nucleic acid molecule in a random coil orother non-structured state. Nucleic acid nanostructures may includedeoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleicacid (PNA), other nucleic acid analogs, and combinations thereof.Nucleic acid nanostructures may have naturally-arising or engineeredsecondary, tertiary, or quaternary structures. A structured nucleic acidparticle can contain at least one of: i) a moiety that is configured tocouple an analyte to the nucleic acid nanostructure, ii) a moiety thatis configured to couple the nucleic acid nanostructure to another objectsuch as another SNAP, a solid support or a surface thereof, iii) amoiety that is configured to provide a chemical or physical property orcharacteristic to a nucleic acid nanostructure, or iv) a combinationthereof. Exemplary SNAPs may include nucleic acid nanoballs (e.g. DNAnanoballs), nucleic acid nanotubes (e.g. DNA nanotubes), and nucleicacid origami (e.g. DNA origami). A SNAP may be functionalized to includeone or more reactive handles or other moieties. A SNAP may comprise oneor more incorporated residues that contain reactive handles or othermoieties (e.g., modified nucleotides).

As used herein, the term “primary structure,” when used in reference toa nucleic acid, refers to a residue sequence of a single-strandednucleic acid. As used herein, the term “secondary structure,” when usedin reference to a nucleic acid, refers to the base-pairing interactionswithin a single nucleic acid polymer or between two polymers. Secondarystructure may include multi-stranded nucleic acids formed byself-complementarity of a single oligonucleotide, such as stems, loops,bulges, and junctions. As used herein, the term “tertiary structure,”when used in reference to a nucleic acid, refers to thethree-dimensional conformation of a nucleic acid, such as the overallthree-dimensional shape of a single-stranded nucleic acid ormulti-stranded nucleic acid.

As used herein, the term “pervious,” when used in reference to astructure of a nucleic acid, refers to the structure containing two ormore structural elements (e.g., single-stranded nucleic acids,double-stranded nucleic acids, a nucleic acid strand containingdouble-stranded and single-stranded nucleic acids, non-nucleic acidmoieties, etc.) having a spatial degree of freedom (e.g., translational,rotational, vibrational, bending, etc.) to facilitate contact of the twoor more structural elements with another molecule. The other moleculecan be, for example, a molecule having a molecular weight greater than0.5, 1, 5, 10 or more kiloDaltons. Optionally, each structural elementof the two or more structural elements can move in concert with themovement of the nucleic acid. Optionally, for an unbound nucleic acidcomprising a pervious structure containing a plurality of pendant,non-interacting moieties, each pendant moiety will rotate if the nucleicacid rotates, but a free terminus of each pendant moiety is capable ofmoving independently of the motion of the other free termini of theother pendant moieties. A spatial degree of freedom may be assessed fora structural element of a nucleic acid with respect to a natural and/orstochastic spatial variation in the structure of the nucleic acid (e.g,a spatial degree of freedom comprising motion beyond the natural thermalor Brownian motion of the nucleic acid structure). A first structuralelement of a pervious structure may have a spatial degree of freedomwith respect to a second structural element in one spatial dimension,two spatial dimensions, or three spatial dimensions. A perviousstructure may be characterized as comprising a differing chemicalcharacteristic from a compacted structure of a nucleic acid, as setforth herein, such as greater or lesser mass diffusivity for smallmolecules or macromolecules, a greater or lesser hydrophobicity, agreater or lesser hydrophilicity, a greater or lesser binding strengthor specificity for another nucleic acid, a greater or lesser likelihoodof binding another nucleic acid, a greater or lesser likelihood ofbinding a solid support, a greater or lesser binding strength orspecificity for a solid support, or a combination thereof. A perviousstructure may comprise a differing characteristic or configuration whenbound to another entity (e.g., a solid support, a second nucleic acid).In some configurations, when bound to a second entity, a perviousstructure may satisfy one or more of: i) each structural element of thetwo or more structural elements moving in concert with a movement of thenucleic acid, ii) each structural element of the two or more structuralelements having a reduced spatial degree of freedom relative to anunbound configuration, and iii) each structural element of the two ormore structural elements containing at least one spatial degree offreedom (e.g., translational, rotational, vibrational, bending, etc.)with respect to each other structural element of the two or morestructural elements. For example, for a nucleic acid coupled to a solidsupport by a pervious structure containing a plurality of pendant,non-interacting moieties, each pendant moiety may be coupled to acomplementary moiety on the solid support, thereby co-locating thenucleic acid and its pervious structure on the solid support, but eachpendant moiety may possess an independent ability to disrupt an existinginteraction with a complementary surface moiety and form a newinteraction with a differing complementary surface moiety.

As used herein, the term “residue,” when used in reference to a polymer,refers to a monomeric unit of a polymer structure. When used inreference to a nucleic acid, a residue may refer to a nucleotide,nucleoside, or a synthetic, modified, or non-natural analogue thereof.When used in reference to a polypeptide, a residue may refer to an aminoacid or a synthetic, modified, or non-natural analogue thereof.

As used herein, the terms “type” or “species,” when used in reference toa molecule, refer to a molecule with a unique, distinguishable chemicalstructure. As used herein, the term “type of SNAP” refers to a SNAP witha unique, distinguishable primary structure, for example, compared toother SNAPs. Two SNAPs are of the same species if they possess the sameprimary, secondary or tertiary structure. SNAP variants are differentspecies from each other. For example, members of a “type of SNAP” canhave a unique, distinguishable structure that is common to the memberscompared to other SNAPs that lack the unique, distinguishable structure.SNAP types may be identified, for example, by common shape and/orconformation, number of coupling sites, or type of coupling sites.

As used herein, the terms “click reaction,” “click-type reaction,” or“bioorthogonal reaction” refer to single-step,thermodynamically-favorable conjugation reaction utilizing biocompatiblereagents. A click reaction may be configured to not utilize toxic orbiologically incompatible reagents (e.g., acids, bases, heavy metals) orto not generate toxic or biologically incompatible byproducts. A clickreaction may utilize an aqueous solvent or buffer (e.g., phosphatebuffer solution, Tris buffer, saline buffer, MOPS, etc.). A clickreaction may be thermodynamically favorable if it has a negative Gibbsfree energy of reaction, for example a Gibbs free energy of reaction ofless than about −5 kiloJoules/mole (kJ/mol), −10 kJ/mol, −25 kJ/mol, −50kJ/mol, −100 kJ/mol, −200 kJ/mol, −300 kJ/mol, −400 kJ/mol, or less than−500 kJ/mol. Exemplary bioorthogonal and click reactions are describedin detail in WO 2019/195633A1, which is herein incorporated by referencein its entirety. Exemplary click reactions may include metal-catalyzedazide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition,strain-promoted azide-nitrone cycloaddition, strained alkene reactions,thiol-ene reaction, Diels-Alder reaction, inverse electron demandDiels-Alder reaction, [3+2] cycloaddition, [4+1] cycloaddition,nucleophilic substitution, dihydroxylation, thiol-yne reaction,photoclick, nitrone dipole cycloaddition, norbornene cycloaddition,oxanobornadiene cycloaddition, tetrazine ligation, and tetrazolephotoclick reactions. Exemplary functional groups or reactive handlesutilized to perform click reactions may include alkenes, alkynes,azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides,aziridines, activated esters, and tetrazines. Other well-known clickconjugation reactions may be used having complementary bioorthogonalreaction species, for example, where a first click component comprises ahydrazine moiety and a second click component comprises an aldehyde orketone group, and where the product of such a reaction comprises ahydrazone functional group or equivalent.

As used herein, the term “array” refers to a population of molecules oranalytes that are attached to unique identifiers such that the analytescan be distinguished from each other. As used herein, the term “uniqueidentifier” refers to a solid support (e.g., particle or bead), spatialaddress in an array, tag, label (e.g., luminophore), or barcode (e.g.,nucleic acid barcode) that is attached to an analyte and that isdistinct from other identifiers, throughout one or more steps of aprocess. The process can be an analytical process such as a method fordetecting, identifying, characterizing or quantifying an analyte.Attachment to a unique identifier can be covalent or non-covalent (e.g.,ionic bond, hydrogen bond, van der Waals forces etc.). A uniqueidentifier can be exogenous to the analyte, for example, beingsynthetically attached to the analyte. Alternatively, a uniqueidentifier can be endogenous to the analyte, for example, being attachedor associated with the analyte in the native milieu of the analyte. Anarray can include different analytes that are each attached to differentunique identifiers. For example, an array can include differentmolecules or analytes that are each located at different addresses on asolid support. Alternatively, an array can include separate solidsupports each functioning as an address that bears a different moleculeor analyte, where the different molecules or analytes can be identifiedaccording to the locations of the solid supports on a surface to whichthe solid supports are attached, or according to the locations of thesolid supports in a liquid such as a fluid stream. The molecules oranalytes of the array can be, for example, nucleic acids such as SNAPs,polypeptides, proteins, peptides, oligopeptides, enzymes, ligands, orreceptors such as antibodies, functional fragments of antibodies oraptamers. The addresses of an array can optionally be opticallyobservable and, in some configurations, adjacent addresses can beoptically distinguishable when detected using a method or apparatus setforth herein. As used herein, the terms “address,” “binding site,” and“site,” when used in reference to an array, means a location in an arraywhere a particular molecule or analyte is present. An address cancontain only a single molecule or analyte, or it can contain apopulation of several molecules or analytes of the same species (i.e. anensemble of the molecules). Alternatively, an address can include apopulation of molecules or analytes that are different species.Addresses of an array are typically discrete. The discrete addresses canbe contiguous, or they can have interstitial spaces between each other.An array useful herein can have, for example, addresses that areseparated by less than 100 microns, 10 microns, 1 micron, 500 nm, 100nm, 10 nm or less. Alternatively or additionally, an array can haveaddresses that are separated by at least 10 nm, 100 nm, 500 nm, 1micron, 5 microns, 10 microns, 50 microns, 100 microns or more. Theaddresses can each have an area of less than 1 square millimeter, 500square microns, 100 square microns, 25 square microns, 1 square micronor less. An array can include at least about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁸,1×10¹⁰, 1×10¹², or more addresses.

As used herein, the term “solid support” refers to a substrate that isinsoluble in aqueous liquid. Optionally, the substrate can be rigid. Thesubstrate can be non-porous or porous. The substrate can optionally becapable of taking up a liquid (e.g., due to porosity) but willtypically, but not necessarily, be sufficiently rigid that the substratedoes not swell substantially when taking up the liquid and does notcontract substantially when the liquid is removed by drying. A nonporoussolid support is generally impermeable to liquids or gases. Exemplarysolid supports include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.),nylon, ceramics, resins, Zeonor™, silica or silica-based materialsincluding silicon and modified silicon, carbon, metals, metal oxides(e.g., zirconia, titania, alumina, etc.), inorganic glasses, opticalfiber bundles, gels, and polymers.

As used herein, the terms “group” and “moiety” are intended to besynonymous when used in reference to the structure of a molecule. Theterms refer to a component or part of the molecule. The terms do notnecessarily denote the relative size of the component or part comparedto the rest of the molecule, unless indicated otherwise. A group ormoiety can contain one or more atom. As used herein, the term “displaymoiety” refers to a component or part of a molecule that is configuredto couple the molecule to an analyte or that couples the molecule to theanalyte. As used herein, the term “capture moiety” refers to a componentor part of a molecule that is configured to couple the molecule to asolid support, surface or interface, or that couples the molecule to thesolid support, surface or interface. As used herein, the term “couplingmoiety” refers to a component or part of a molecule that is configuredto couple the molecule to a second molecule, or that couples themolecule to the second molecule. As used herein, the term “utilitymoiety” refers to a component or part of a molecule that is configuredto provide a functionality or structure to the molecule, or thatprovides the functionality or structure to the molecule. Thefunctionality or structure can be a new function or structure that isnot provided by a display moiety, capture moiety, or coupling moiety ofthe molecule; or it can be a modification (e.g., inhibition oractivation) of a structure or function that is provided by a displaymoiety, capture moiety, or coupling moiety of the molecule.

As used herein, the term “face” refers to a portion of a molecule,particle, or complex (e.g., a SNAP or a SNAP complex) that contains oneor more moieties with substantially similar orientation and/or function.For example, a substantially rectangular or square SNAP may have acoupling face that comprises one or more coupling moieties, with eachcoupling moiety having a substantially similar orientation to each othercoupling moiety (e.g., oriented about 180° from a display moiety that isconfigured to be coupled to an analyte). In another example, a sphericalnanoparticle may have a coupling face comprising a coupled plurality ofcoupling moieties confined to a hemisphere of the particle (i.e., aplurality of coupling moieties having similar function but differingorientations). In some cases, a face may be defined by an imaginaryplane relative to which a moiety or a portion thereof may have a spatialproximity or angular orientation when the plane is contacted with apoint or portion of a molecule, particle, or complex. A moiety or aportion thereof may have a spatial separation from an imaginary planedefining a face of a molecule, particle, or complex of no more thanabout 100 nanometers (nm), 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30nm, 25 nm 20 nm, 15 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm,2 nm, 1 nm, 0.5 nm, 0.1 nm, or less than 0.1 nm. A moiety or a portionthereof may have an angular orientation relative to a normal vector ofan imaginary plane of no more than about 90°, 85°, 80°, 75°, 70°, 65°,60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 5°, 1°, or lessthan 1°.

As used herein, the term “analyte” and “analyte of interest,” when usedin reference to a structured nucleic acid particle, refer to a molecule,particle, or complex of molecules or particles that is coupled to adisplay moiety of a structured nucleic acid particle. An analyte maycomprise a target for an analytical method (e.g., sequencing,identification, quantification, etc.) or may comprise a functionalelement such as a binding ligand or a catalyst. An analyte may comprisea biomolecule, such as a polypeptide, polysaccharide, nucleic acid,lipid, metabolite, enzyme cofactor or a combination thereof. An analytemay comprise a non-biological molecule, such as a polymer, metal, metaloxide, ceramic, semiconductor, mineral, or a combination thereof. Asused herein, the terms “sample analyte” refers to an analyte derivedfrom a sample collected from a biological or non-biological system. Asample analyte may be purified or unpurified. As used herein, the term“control analyte” refers to an analyte that is provided as a positive ornegative control for comparison to a sample analyte. A control analytemay be derived from the same source as a sample analyte, or derived froma differing source from the sample analyte. As used herein, the term“standard analyte” refers to a known or characterized analyte that isprovided as a physical or chemical reference to a process. A standardanalyte may comprise the same type of analyte as a sample analyte, ormay differ from a sample analyte. For example, a polypeptide analyteprocess may utilize a polypeptide standard analyte with knowncharacteristics. In another example, a polypeptide analyte process mayutilize a non-polypeptide standard analyte with known characteristics.As used herein, the term “inert analyte” refers to an analyte with noexpected function in a process or system.

As used herein, the terms “linker,” “linking group,” or “linking moiety”refer to a molecule or molecular chain that is configured to attach afirst molecule to a second molecule. A linker, linking group, or linkingmoiety may be configured to provide a chemical or mechanical property toa region separating a first molecule from a second molecule, such ashydrophobicity, hydrophilicity, electrical charge, polarity, rigidity,or flexibility. A linker, linking group, or linking moiety may comprisetwo or more functional groups that facilitate the coupling of thelinker, linking group, or linking moiety to the first and secondmolecule. A linker, linking group, or linking moiety may includepolyfunctional linkers such as homobifunctional linkers,heterobifunctional linkers, homopolyfunctional linkers, andheteropolyfunctional linkers. The molecular chain may be characterizedby a minimum size such as, for example, at least about 100 Daltons (Da),200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1kiloDalton (kDa), 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa ormore than 20 kDa. Alternatively or additionally, a molecular chain maybe characterized by a maximum size such as, for example, no more thanabout 20 kDa, 15 kDa, 10 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, 900 Da,800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, 100 Da, or lessthan 100 Da. Exemplary molecular chains may comprise polyethylene glycol(PEG), polyethylene oxide (PEO), alkane chains, fluorinated alkanechains, dextrans, and polynucleotides.

As used herein, the terms “reversible” and “reversibility” are used inreference to a chemical or physical coupling of two entities (e.g.,molecules, analytes, functional groups, or moieties) that has asubstantial likelihood of uncoupling under one or more conditions ofuse. Reversibility may consist of thermodynamic reversibility, kineticreversibility, or a combination thereof. Reversible coupling of a firstentity to a second entity may be characterized by a temporary change tothe structure or function of the first and/or second entity when coupledto each other. Reversing the coupling can optionally revert thestructure or function of the first and/or second entity to the samestate as it was prior to the temporary change. The context fordetermining reversibility may comprise the likelihood of detecting areversed coupling given the specific spatial, temporal, and physicalenvironment in which two coupled molecules are located. For example, ina population of one million streptavidin-biotin coupled pairs, adetectable number of reversed couplings may be predictedthermodynamically, however the slow kinetic reversal of the bindingreaction may make such decouplings not detectable above detection noiseif the detection time scale is on the order of seconds or minutes. Inthis context, the streptavidin-biotin coupling would be described asirreversible. The context of reversibility may be process-dependent fora system that undergoes multiple processes. For example, measurablede-coupling of coupled molecules may occur during months of storage buta subsequent process utilizing the coupled molecules may occur inminutes. In this context, the coupled molecules may be reversiblycoupled with respect to storage but irreversibly coupled with respect toutilization. Measures of reversibility may include use of quantitativemeasures such as equilibrium constants or kinetic on-rates and/oroff-rates. Reversibility may be directly measured by an equilibriumassay. Reversibility may vary with changes in a chemical system, such aschanges in temperature or solvent composition. A reversible coupling mayinclude meta-stable couplings that remain coupled until a change inphysical environment. For example, complementary nucleic acids mayremain stably coupled at 20° C. but may rapidly decouple above 75° C. Areversible coupling may remain coupled for a time period of at leastabout 1 second (s), 1 minute (min), 5 min, 10 min, 15 min, 30 min, 1hour (hr), 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 12 hr, 18 hr, 1 day, 1 week, 1month, 6 months, 1 year, or more than 1 year. Alternatively oradditionally, a reversible coupling may become decoupled in a timeperiod of no more than about 1 year, 6 months, 1 month, 1 week, 1 day,18 hrs, 12 hrs, 6 hrs, 5 hrs, 4 hrs, 3 hrs, 2 hrs, 1 hr, 30 min, 15 min,10 min, 5 min, 1 min, 1 s, or less than 1 s.

As used herein, terms “irreversible” and “irreversibility” are used inreference to a chemical or physical coupling of two entities (e.g.,molecules, analytes, functional groups, or moieties) that has alikelihood of remaining coupled under one or more conditions of use. Asystem that is determined to not be reversible as described above may bedescribed as irreversible. For example, irreversible coupling of a firstentity to a second entity may be characterized by a permanent change tothe structure or function of the first and/or second entity after beingcoupled to each other. Uncoupling can cause substantial change to thestructure or function of one or both of the entities compared to thestructure or function of the respective entity or entities prior to thecoupling. An irreversible coupling may remain coupled for a time periodof at least about 1 second (s), 1 minute (min), 5 min, 10 min, 15 min,30 min, 1 hour (hr), 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 12 hr, 18 hr, 1 day,1 week, 1 month, 6 months, 1 year, or more than 1 year.

As used herein, the term “affinity reagent” refers to a molecule orother substance that is capable of specifically or reproducibly bindingto a binding partner or other substance. Binding can optionally be usedto identify, track, capture, alter, or influence the binding partner.The binding partner can optionally be larger than, smaller than or thesame size as the affinity reagent. An affinity reagent may form areversible or irreversible interaction with a binding partner. Anaffinity reagent may bind with a binding partner in a covalent ornon-covalent manner. An affinity reagent may be configured to perform achemical modification (e.g., ligation, cleavage, concatenation, etc.)that produces a detectable change in the larger molecule, therebypermitting observation of the interaction that occurred. Affinityreagents may include chemically reactive affinity reagents (e.g.,kinases, ligases, proteases, nucleases, etc.) and chemicallynon-reactive affinity reagents (e.g., antibodies, antibody fragments,aptamers, DARPins, peptamers, etc.). An affinity reagent may compriseone or more known and/or characterized binding components or bindingsites (e.g., complementarity-defining regions) that mediate orfacilitate binding with a binding partner. Accordingly, an affinityreagent can be monovalent or multivalent (e.g. bivalent, trivalent,tetravalent, etc.). An affinity reagent is typically non-reactive andnon-catalytic, thereby not permanently altering the chemical structureof a substance it binds in a method set forth herein.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably to refer to a molecule or analyte comprising two or moreamino acids joined by a peptide bond. A polypeptide may refer to apeptide (e.g., a polypeptide with less than about 200, 150, 100, 75, 50,40, 30, 20, 15, 10, or less than about 10 linked amino acids). Apolypeptide may refer to a naturally-occurring molecule, or anartificial or synthetic molecule. A polypeptide may include one or morenon-natural, modified amino acids, or non-amino acid linkers. Apolypeptide may contain D-amino acid enantiomers, L-amino acidenantiomers or both. A polypeptide may be modified naturally orsynthetically, such as by post-translational modifications.

As used herein, the term “detectable label” refers to a moiety of anaffinity reagent or other substance that provides a detectablecharacteristic. The detectable characteristic can be, for example, anoptical signal such as absorbance of radiation, luminescence orfluorescence emission, luminescence or fluorescence lifetime,luminescence or fluorescence polarization, or the like; Rayleigh and/orMie scattering; binding affinity for a ligand or receptor; magneticproperties; electrical properties; charge; mass; radioactivity or thelike. A label component can be a detectable chemical entity that isconjugated to or capable of being conjugated to another molecule orsubstance. Exemplary molecules that can be conjugated to a labelcomponent include an affinity reagent or a binding partner. A labelcomponent may produce a signal that is detected in real-time (e.g.,fluorescence, luminescence, radioactivity). A label component mayproduce a signal that is detected off-line (e.g., a nucleic acidbarcode) or in a time-resolved manner (e.g., time-resolvedfluorescence). A label component may produce a signal with acharacteristic frequency, intensity, polarity, duration, wavelength,sequence, or fingerprint. Exemplary labels include, without limitation,a fluorophore, luminophore, chromophore, nanoparticle (e.g., gold,silver, carbon nanotubes), heavy atom, radioactive isotope, mass label,charge label, spin label, receptor, ligand, nucleic acid barcode,polypeptide barcode, polysaccharide barcode, or the like.

As used herein, the term “nucleic acid origami” refers to a nucleic acidconstruct comprising an engineered secondary, tertiary or quaternarystructure. A nucleic acid origami may include DNA, RNA, PNA, LNAs, othernucleic acid analog, modified or non-natural nucleic acids, orcombinations thereof. A nucleic acid origami may comprise a plurality ofoligonucleotides that hybridize via sequence complementarity to producethe engineered structuring of the origami particle. A nucleic acidorigami may comprise sections of single-stranded or double-strandednucleic acid, or combinations thereof. A nucleic acid origami maycomprise one or more tertiary structures of a nucleic acid, such asA-DNA, B-DNA, C-DNA, L-DNA, M-DNA, Z-DNA, etc. A nucleic acid origamimay comprise single-stranded nucleic acid, double-stranded nucleic acid,multi-stranded nucleic acid, or combinations thereof. Exemplary nucleicacid origami structures may include nanotubes, nanowires, cages, tiles,nanospheres, blocks, and combinations thereof.

As used herein, the term “nucleic acid nanoball” refers to a globular orspherical nucleic acid structure. A nucleic acid nanoball may comprise aconcatemer of oligonucleotides that arranges in a globular structure. Anucleic acid nanoball may comprise one or more oligonucleotides,including oligonucleotides comprising self-complementary nucleic acidsequences. A nucleic acid nanoball may comprise a palindromic nucleicacid sequence. A nucleic acid nanoball may include DNA, RNA, PNA, LNAs,other nucleic acid analog, modified or non-natural nucleic acids, orcombinations thereof.

As used herein, the term “oligonucleotide” refers to a moleculecomprising two or more nucleotides joined by a phosphodiester bond oranalog thereof. An oligonucleotide may comprise DNA, RNA, PNA, LNAs,other nucleic acid analog, modified nucleotides, non-naturalnucleotides, or combinations thereof. An oligonucleotide may include alimited number of bonded nucleotides, such as, for example, less thanabout 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70,60, 50, 40, 30, 25, 20, 15, 10, or less than 5 nucleotides. Anoligonucleotide may include a linking group or linking moiety at aterminal or intermediate position. For example, an oligonucleotide maycomprise two nucleic acid strands that are joined by an intermediate PEGmolecule. In another example, an oligonucleotide may comprise acleavable linker (e.g., a photocleavable linker, anenzymatically-cleavable linker, a restriction site, etc.) that joins twoportions of the oligonucleotide. The terms “polynucleotide” and “nucleicacid” are used herein synonymously with the term “oligonucleotide.”

As used herein, the term “scaffold” refers to a molecule or complex ofmolecules having a structure that couples two or more entities to eachother. A scaffold can form a structural basis for coupling bindingcomponents and/or labeling components to a detectable probe. A scaffoldmay comprise a plurality of attachment sites that permit the coupling orconjugation of detectable probe components to the scaffold. Scaffoldattachment sites may include functional groups, active sites, bindingligands, binding receptors, nucleic acid sequences, or any other entitycapable of forming a covalent or non-covalent attachment to a bindingcomponent, label component, or other detectable probe component. Ascaffold may comprise an oligonucleotide molecule that serves as theprimary structural unit for a nucleic acid origami. A scaffold maycomprise single-stranded nucleic acids, double-stranded nucleic acids,or combinations thereof. A scaffold may be a circular oligonucleotide ora linear (i.e. non-circular) oligonucleotide. A scaffold may be derivedfrom a natural source, such as a bacterial or viral genome (e.g.,plasmid DNA or a phage genome). A circular scaffold may be formed by theligation of a non-circular nucleic acid. A scaffold may comprise aparticular number of nucleotides, for example, at least about 500, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000,7500, 8000, 8500, 9000, 9500, 10000, or more than 10000oligonucleotides. A scaffold may comprise an organic or inorganicparticle or nanoparticle. A scaffold may comprise a coating or layerapplied to a particle or nanoparticle that permits attachment ofdetectable label components.

As used herein, the term “two-dimensional projection” refers to the areaor shape that would be occupied by the projection of a three-dimensionalstructure onto a planar two-dimensional surface without substantialgeometric or spatial distortion. For example, the two-dimensionalprojection of a sphere onto a planar two-dimensional surface wouldproduce a circular area on the surface with a diameter equivalent to thediameter of the sphere. A two-dimensional projection may be formed fromany frame of reference, including a frame of reference that isorthogonal to any surface of the three-dimensional structure. Manythree-dimensional structures are capable of producing projections ofdifferent size or shape depending upon the frame of reference.Accordingly, the largest two-dimensional projection for athree-dimensional structure refers to the largest area or shape that isproduced from all frames of reference for the three-dimensionalstructure; the smallest two-dimensional projection for athree-dimensional structure refers to the smallest area or shape that isproduced from all frames of reference for the three-dimensionalstructure; and the average two-dimensional projection for athree-dimensional structure refers to the average area or shape that isproduced from all frames of reference for the three-dimensionalstructure.

As used herein, the term “effective surface area,” when used inreference to a nucleic acid, refers to a surface area of atwo-dimensional projection of the nucleic acid or a portion thereof whenthe nucleic acid is not bound to a surface (e.g., solvated or suspendedin a fluidic medium). As used herein, the term “footprint,” when used inreference to a nucleic acid, refers to a surface area of atwo-dimensional projection of the nucleic acid or a portion thereof whenthe nucleic acid is bound to a surface (e.g., coupled to a solidsupport). FIG. 48 depicts a difference between an effective surface areaand a footprint of a nucleic acid. In an unbound configuration, atwo-dimensional projection of the nucleic acid 4810 onto a surface 4800would have a surface area that is proportional to a length, l₁, that issubstantially the same as a distance between the two ends of the unboundnucleic acid 4810. In a bound configuration, the coupling of the nucleicacid 4810 to the surface 4800 increases the distance between the ends ofthe nucleic acid, thereby increasing the surface area of thetwo-dimensional projection of the nucleic acid onto the surface 4800.Accordingly, the nucleic acid has a larger footprint than its effectivesurface area.

As used herein, the term “offset” refers to the spatial difference inorientation between two lines (2-dimensional) or surfaces(3-dimensional). An offset may include a distance offset and/or anangular offset. FIGS. 1A and 1B depict examples of angular offset fordiffering two-dimensional shapes (which could be two-dimensionalprojections of three-dimensional structures). The isosceles triangle 100of FIG. 1A has an angular offset of 120° between the first face 110 andthe second face 120 whose relative orientations are depicted byorthogonal vectors A and A′. The rectangle 130 of FIG. 1B has an angularoffset of 180° between the first face 110 and the second face 120, whoserelative orientations are depicted by orthogonal vectors A and A′.

As used herein, the term “binding specificity” refers to the tendency ofan affinity reagent to preferentially interact with a binding partner,affinity target, or target moiety relative to other binding partners,affinity targets, or target moieties. An affinity reagent may have acalculated, observed, known, or predicted binding specificity for anypossible binding partner, affinity target, or target moiety. Bindingspecificity may refer to selectivity for a single binding partner,affinity target, or target moiety in a sample over at least one otheranalyte in the sample. Moreover, binding specificity may refer toselectivity for a subset of binding partners, affinity targets, ortarget moieties in a sample over at least one other analyte in thesample.

As used herein, the term “binding affinity” or “affinity” refers to thestrength or extent of binding between an affinity reagent and a bindingpartner, affinity target or target moiety. In some cases, the bindingaffinity of an affinity reagent for a binding partner, affinity target,or target moiety may be vanishingly small or effectively zero. A bindingaffinity of an affinity reagent for a binding partner, affinity target,or target moiety may be qualified as being a “high affinity,” “mediumaffinity,” or “low affinity.” A binding affinity-of an affinity reagentfor a binding partner, affinity target, or target moiety may bequantified as being “high affinity” if the interaction has adissociation constant of less than about 100 nM, “medium affinity” ifthe interaction has a dissociation constant between about 100 nM and 1mM, and “low affinity” if the interaction has a dissociation constant ofgreater than about 1 mM. Binding affinity-can be described in termsknown in the art of biochemistry such as equilibrium dissociationconstant (K_(D)), equilibrium association constant (K_(A)), associationrate constant (k_(on)), dissociation rate constant (k_(off)) and thelike. See, for example, Segel, Enzyme Kinetics John Wiley and Sons, NewYork (1975), which is incorporated herein by reference in its entirety.

As used herein, the term “promiscuity,” when used in reference tobinding, may refer to affinity reagent properties of 1) binding to aplurality of binding partners due to the presence of a particularaffinity target or target moiety, regardless of the binding context ofthe affinity target or target moiety; or 2) binding to a plurality ofaffinity targets or target moieties within the same or differing bindingpartners; or 3) a combination of both properties. With regard to thefirst form of binding promiscuity, “binding context” may refer to thelocal chemical environment surrounding an affinity target or targetmoiety, such as flanking, adjacent, or neighboring chemical entities(e.g., for a polypeptide epitope, flanking amino acid sequences, oradjacent or neighboring non-contiguous amino acid sequences relative tothe epitope). With regard to the second form of binding promiscuity, thedefinition may refer to an affinity reagent or probe binding tostructurally- or chemically-related affinity targets or target moietiesdespite differences between the affinity targets or target moieties. Forexample, an affinity reagent may be considered promiscuous if itpossesses a binding affinity for trimer peptide sequences having theform WXK, where W is tryptophan, K is lysine and X is any possible aminoacid. Additional concepts pertaining to binding promiscuity arediscussed in WO 2020106889A1, which is incorporated herein by referencein its entirety.

As used herein, the term “binding probability” refers to the probabilitythat an affinity reagent may be observed to interact with a bindingpartner and/or an affinity target within a particular binding context. Abinding probability may be expressed as a discrete number such as avalue N in the range 0≤N≤1 (e.g. 0.4) or a percent value (e.g., 40%), amatrix of discrete ambers, or as mathematical model (e.g., a theoreticalor empirical model). A binding probability may include one or morefactors, including the binding specificity, the likelihood of locatingthe affinity target, and the likelihood of binding for a sufficientamount of time for the binding interaction to be detected. An overallbinding probability may include binding probability when all factorshave been weighted relative to the binding context.

As used herein, the term “binding context” may refer to theenvironmental conditions in which an affinity reagent-binding partnerinteraction is observed. The binding context may be a constant conditionor a condition that changes within a range. Environmental conditions mayinclude any factors that may influence an interaction between anaffinity reagent and a binding partner, such as temperature, fluidproperties (e.g., ionic strength, polarity, pH), relativeconcentrations, absolute concentrations, fluid composition, bindingpartner conformation, affinity reagent conformation, and combinationsthereof.

As used herein the term “tunable”, when used in reference to astructured nucleic acid particle, refers to the specific, precise,and/or rational location of components or attachment sites forcomponents with an assembly or structure. Tunable retaining componentsmay refer to the ability to couple or conjugate probe components atspecific sites or within specific regions of the retaining componentstructure, or to generate attachment sites for the coupling orconjugation of probe components at specific sites or specific regions ofthe retaining component structure. As used herein, “tunability” refersto the property of a probe or retaining component having a tunablestructure or architecture.

As used herein, the term “functional group” refers to a group of atomsin a molecule that confer a chemical property, such as reactivity,polarity, hydrophobicity, hydrophilicity, solubility, etc., on themolecule. Functional groups may comprise organic moieties or maycomprise inorganic atoms. Exemplary functional groups may include alkyl,alkenyl, alkynyl, phenyl, halide, hydroxyl, carbonyl, aldehyde, acylhalide, ester, carboxylate, carboxyl, carboalkoxy, methoxy,31ydroperoxyl, ether, hemiacetal, hemiketal, acetal, ketal, orthoester,epoxide, carboxylic anhydride, carboxamide, amine, ketimine, aldimine,imide, azide, azo, cyanate, isocyanate, nitrate, nitrile, isonitrile,nitrosoxy, nitro, nitroso, oxime, pyridyl, carbamate, sulfhydryl,sulfide, disulfide, sulfinyl, sulfonyl, sulfinom, sulfo, thiocyanate,isothiocyanate, carbonothioyl, thioester, thionoester, phosphino,phosphono, phosphonate, phosphate, borono, boronate, and borinatefunctional groups.

As used herein, the term “functionalized” refers to any material orsubstance that has been modified to include a functional group. Afunctionalized material or substance may be naturally or syntheticallyfunctionalized. For example, a polypeptide can be naturallyfunctionalized with a phosphate, oligosaccharide (e.g., glycosyl,glycosylphosphatidylinositol or phosphoglycosyl), nitrosyl, methyl,acetyl, lipid (e.g., glycosyl phosphatidylinositol, myristoyl orprenyl), ubiquitin or other naturally occurring post-translationalmodification. A functionalized material or substance may befunctionalized for any given purpose, including altering chemicalproperties (e.g., altering hydrophobicity or changing surface chargedensity) or altering reactivity (e.g., capable of reacting with a moietyor reagent to form a covalent bond to the moiety or reagent).

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” As used herein, the term “about,” when used in connection withpercentages, may mean a variance of at most ±5% of the value beingreferred to. For example, about 90% may mean from 85% to 95%. In somecases, “about” may mean a valiance of at most ±4%, ±3%, ±2%, ±1%, ±0.5%or less of the value being referred to. As used herein, the term“substantially,” when used in reference to a measurable quantity orproperty, refers to the quantity or property having a value within ±10%of a reference value. For example, a first value may be substantiallythe same as a second value if the first value is within ±10% of thesecond value. In another example, a shape may be substantially square ifa ratio of side lengths of a rectangle is within a range between 0.90and 1.10, inclusive. In some cases, “substantially” may mean a quantityor property having a value within at most ±9%, ±8%, ±7%, ±6%, ±5%, ±4%,±3%, ±2%, ±1%, ±0.5%, or less of a reference value.

As used herein, the terms “attached” or “coupled” refer to the state oftwo things being joined, fastened, adhered, connected or bound to eachother. Attachment can be covalent or non-covalent. For example, aparticle can be attached or coupled to a protein by a covalent ornon-covalent bond. Similarly, a first nucleic acid can be attached orcoupled to a second nucleic acid via hybridization or Watson-Crick basepairing. A covalent bond is characterized by the sharing of pairs ofelectrons between atoms. A non-covalent bond is a chemical bond thatdoes not involve the sharing of pairs of electrons and can include, forexample, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilicinteractions, adhesion, adsorption, and hydrophobic interactions.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

Nucleic Acid Structures

Provided herein are nucleic acids that are useful for the formation ofarrays of analytes that permit the interrogation of the analytes of thearray at single-analyte resolution. The nucleic acids set forth hereincan be characterized as possessing tunable two-dimensional orthree-dimensional structures that facilitate one or more characteristicsselected from: i) displaying an analyte in an orientation thatfacilitates interrogation of the analyte at single-analyte resolution;ii) maximizing likelihood of coupling to a solid support or a surfacethereof at a site that is configured to bind the nucleic acid; iii)maximizing likelihood of coupling to a site on a solid support orsurface thereof in a controllable and/or non-random fashion; iv)minimizing a likelihood of coupling to a solid support or a surfacethereof at a site that is already occupied by another nucleic acid; andv) minimizing a likelihood of coupling to a solid support or a surfacethereof at an address that is not configured to bind the nucleic acid.In some configurations, a nucleic acid, as set forth herein, may possessall of the aforementioned characteristics. In other configurations, twoor more nucleic acids may be complexed, in which the nucleic acidcomplex possesses all of the aforementioned characteristics.

Described herein are nucleic acids that are useful for the organizationof individual moieties in single-analyte systems. A nucleic acid, as setforth herein, may be characterized by one or more characteristics of: i)comprising a display moiety that is configured to couple an analyte tothe nucleic acid, or that couples the analyte to the nucleic acid; ii)comprising a capture moiety that is configured to couple the nucleicacid to a solid support or a surface thereof, or that couples thenucleic acid to the solid support or surface thereof; iii) comprising acoupling moiety that is configured to couple a second molecule to thenucleic acid, or that couples the second molecule to the nucleic acid;and iv) comprising a utility moiety that modifies a physical and/orchemical property of the nucleic acid. In some cases, the nucleic acidis a nucleic acid nanostructure or structured nucleic acid particle(SNAP).

A nucleic acid, as set forth herein, may comprise a naturally-occurringnucleic acid structure, such as a naturally-occurring primary structure(e.g., a naturally-occurring single-stranded nucleotide sequence, asingle strand of a plasmid, etc.), a naturally-occurring secondarystructure (e.g., a naturally-occurring A-DNA, B-DNA, Z-DNA ordouble-stranded helical structure), a naturally-occurring tertiarystructure (e.g., a nucleic acid comprising an origami structurenucleosome, chromatin, etc.). A nucleic acid, as set forth herein, maycomprise a synthetic, artificial, or engineered nucleic acid structure.In some configurations, a nucleic acid may comprise a nucleic acidnanostructure, in which the nucleic acid nanostructure comprises acompacted three-dimensional structure. A nucleic acid nanostructure maycomprise one or more structures that are not known to occur in anaturally-occurring nucleic acid. A nucleic acid nanostructure maycomprise one or more structures with a characterizable property thatdiffers from the same characterizable property of a naturally-occurringnucleic acid (e.g., a higher or lower average persistence length over anucleic acid strand comprising N nucleotides, a higher or lower radiusof curvature of a nucleic acid strand comprising at least 75%double-stranded nucleic acid, a shorter or longer distance between twonon-contiguous regions of a nucleic acid strand, a temporal variation inany aforementioned property, etc.).

The compositions and methods set forth herein will generally beexemplified with reference to a nucleic acid nanostructure or SNAP;however, it will be understood that the methods and compositionsexemplified can be extended to other nucleic acids, such as those setforth herein.

It will also be understood that the nucleic acid structures aredescribed with respect to an average spatial and/or temporalconfiguration. A nucleic acid structure, as set forth herein, can be ina dynamic state with respect to common physical phenomena (e.g., thermalmotion, intermolecular collisions, externally-applied forces,intramolecular vibration, intramolecular bending, intramolecularrotation, etc.) that cause spatial and/or temporal variations in theconfiguration of the nucleic acid. Quantitative descriptions of nucleicacid structure can include spatial and/or temporal variations inaccordance with the dynamic nature of molecular structure understood inthe art.

Aspects of Nucleic Acid Structure: A nucleic acid nanostructure, such asa SNAP, may comprise various structures or structural motifs that giverise to higher ordered structures or geometries. For example, aconcatemerized rolling-circle amplification (RCA) product may produce aglobular nanoball structure with spike-like structures at the outerboundary where the single-stranded, concatemerized nucleic acid formsnearly 180° turns (i.e., a nanoscale urchin-like structure). In anotherexample, a SNAP may comprise a DNA origami particle comprising ascaffold single-stranded nucleic acid hybridized with a plurality ofoligonucleotides that shape the scaffold strand into an overall tertiarystructure. Regions of the tertiary structure may be connected by certainoligonucleotides of the plurality of oligonucleotides to pattern thescaffold into a regular or irregular shapes such as a tile, disc,triangle, torus, cube, pyramid, cylinder, tube, and other more complextwo-dimensional or three-dimensional structures.

A nucleic acid nanostructure, such as a SNAP, may comprise one or morefaces that provide a structural feature and/or perform a function forthe nucleic acid nanostructure. A nucleic acid nanostructure, such as aSNAP, may comprise one or more of: 1) a display face; 2) a capture face;3) a coupling face; and 4) a utility face. A display face may comprise acapture moiety that couples, or is configured to couple, a nucleic acidnanostructure to an analyte. A capture face may comprise a capturemoiety that couples, or is configured to couple, a nucleic acidnanostructure to a surface or interface. A coupling face may comprise acoupling moiety that couples, or is configured to couple, a firstnucleic acid nanostructure to a second nucleic acid nanostructure. Autility face may comprise a utility moiety that provides an additionalutility to a nucleic acid nanostructure (e.g., a SNAP), such asproviding structure, providing stability, altering an interaction (e.g.,attraction or repulsion, steric hindrance, etc.) between a nucleic acidnanostructure and another entity (e.g., a second nucleic acidnanostructure, a surface, etc.), or altering a physical property of anucleic acid nanostructure (e.g., a utility moiety may comprise anelectrical, magnetic, or optical material, etc.). A nucleic acidnanostructure, such as a SNAP, may comprise a face with more than onefunction. For example, a coupling face may also comprise a utility face.In another example, a display face may also comprise a utility face or acapture face. A nucleic acid nanostructure, such as a SNAP, may comprisea face that is comprised of one or more other types of faces. Forexample, a display face may comprise portions or regions that areutility faces comprising steric blocking groups (e.g., PEG, PEO,dextrans, etc.). In some configurations, a multi-function face may becounted as a single face. For example, a cube-like SNAP may compriseabout six distinct faces, with each of the six faces comprising one ormore functions, e.g., a display face and a utility face on one of thesix sides.

A nucleic acid nanostructure, such as a SNAP, may comprise one or morefaces that provide functionality to the nucleic acid nanostructure. Aface may comprise a side or portion of a nucleic acid nanostructure witha similar orientation or two-dimensional projection onto an imaginaryplanar surface. FIG. 2A-2D depict examples of faces for simplifiedstructures similar to those that might be encountered on nanostructuressuch as SNAPs. FIG. 2A shows two shorter tertiary structures 210 and 212(e.g., DNA double helices) linked by a first turning linker 215. The twoshorter tertiary structures 210 and 212 are linked to longer tertiarystructures 220 and 222, which are linked by a third turning linker 225.The two shorter tertiary structures 210 and 212 are linked to the twolonger tertiary structures 220 and 222 by a second turning linker 230.The two shorter tertiary structures 210 and 212 and the two longertertiary structures 220 and 222 are oriented to be coplanar. Functionalgroups R₁, R₂, R₃, and R₄ extend outward from the tertiary structures inparticular orientations that extend out from the plane in which thetertiary structures are oriented. An imaginary plane P is placedorthogonal to, and is intersected by, the four tertiary structures. FIG.2B depicts a cross-sectional view of the tertiary structures taken atplane P. The relative positions of functional groups R₁, R₂, R₃, and R₄are shown with respect to the tertiary structures from which thefunctional groups are displayed. The structures depicted in FIG. 2A canbe defined by four faces, S₁, S₂, T, and B, as shown in FIG. 2B. Thefaces represent a projection of the tertiary structures onto theimaginary planes defined by faces S₁, S₂, B, and T. Due to some degreesof freedom in the position of functional groups and/or moieties that mayextend from the tertiary structures, as well as the size and length ofthe functional groups or moieties, the faces may extend beyond a simpleorthogonal projection of the tertiary structures onto faces S₁, S₂, B,or T. In some cases, a functional group or moiety extending from anucleic acid nanostructure may be considered to be located in two ormore faces of the nucleic acid nanostructure. In other cases, afunctional group or moiety extending from a nucleic acid nanostructuremay be considered to be located within a single face of the nucleic acidnanostructure. The face to which a functional group or moiety isassigned may be defined by the utility or purpose of the functionalgroup or moiety. For example, a moiety with a rigid chain that islocated near two differing faces may be assigned to a single facebecause the orientation caused by the rigid chain makes the moietyfunctionally inaccessible to the other face. Due to the aligned andcoplanar geometry of the tertiary structures, the faces S₁and S₂ wouldorthogonally meet faces B and T if extended. In some cases (e.g., acylindrical or tube structure), a face may comprise up to 360° of totalaspect or orientation.

FIGS. 2C-2D depict the location of nucleic acid nanostructure faces fora plurality of tertiary structures that are not coplanar. FIG. 2C showstwo shorter tertiary structures 210 and 212 (e.g., DNA double helices)linked by a first turning linker 215. The two shorter tertiarystructures 210 and 212 are linked to longer tertiary structures 220 and222, which are linked by a third turning linker 225. The two shortertertiary structures 210 and 212 are linked to the two longer tertiarystructures 220 and 222 by a second turning linker 230. The two shortertertiary structures 210 and 212 are positioned beneath the longertertiary structures 220 and 222. Imaginary, reference plane P′ definesroughly a plane of mirror symmetry with respect to the tertiarystructures. FIG. 2D depicts a projection of the tertiary structures onthe plane P′. Two faces, D and B can be defined for the nucleic acidnanostructure depicted in FIG. 2C. The faces, if extended, wouldintersect, although due to the relative geometry, the intersection wouldnot occur orthogonally.

A nucleic acid nanostructure, such as a SNAP, may have a particularnumber of faces. A nucleic acid nanostructure may have at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore than 20 faces. Additionally or alternatively, a nucleic acidnanostructure may have no more than about 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less than 2 faces. The numberof faces of a nucleic acid nanostructure may be chosen to match afunctionality for the nucleic acid nanostructure. For example, a SNAPthat is configured to couple an analyte to a solid support maynecessitate at least 2 faces (a display face and a coupling face), withadditional faces added based upon other design considerations (e.g.,utility faces).

A nucleic acid nanostructure, such as a SNAP, may comprise two or morefaces where each face has a differing utility. A nucleic acidnanostructure may comprise one or more utilities selected from the groupconsisting of: 1) a display face that couples, or is configured tocouple, an analyte; 2) a capture face that couples, or is configured tocouple, to a surface; 3) a coupling face that couples, or is configuredto couple, a first nucleic acid nanostructure to a second nucleic acidnanostructure; and 4) a utility face that provides any additionalutility (e.g., steric blocking). In some configurations, a nucleic acidnanostructure may comprise a first utility (e.g., a display facecomprising a display moiety) and a second face may comprise a secondutility (e.g., a capture face comprising a capture moiety). In otherconfigurations, two or more faces may have the same utility (e.g., twoor more display faces) but one face of the two or more faces maycomprise a differing utility (e.g., a capture face). In someconfigurations, a nucleic acid nanostructure may comprise the same twoor more utilities on two or more faces (e.g., two opposed faces thatfunction as display faces and capture faces).

A nucleic acid nanostructure, such as a SNAP, may comprise structuralsymmetry, for example, according to an axis of symmetry (i.e.,rotational symmetry) or a plane of symmetry (i.e., reflection symmetry).A tertiary structure of a nucleic acid nanostructure may comprisestructural symmetry, for example, according to an axis of symmetry(e.g., aligned with a centerline of a helical structure). A plurality oftertiary structures taken as a whole may comprise structural symmetry,for example, according to an axis of symmetry or a plane of symmetry. Aface of a nucleic acid nanostructure may be oriented with respect to anaxis or plane of symmetry for the nucleic acid nanostructure or atertiary structure of a plurality of tertiary structures that form thenucleic acid nanostructure. For example, for the cross-section shown inFIG. 2B, the top Face T may be oriented at 0° relative to an axis ofsymmetry that is coaxial to any of the four tertiary structures, whilefaces S₁, B, and S₂, may be oriented at 90°, 180°, and 270°,respectively. For a nucleic acid nanostructure (e.g., a SNAP) comprisinga first tertiary structure and a second tertiary structure, anorientation of a first face (e.g., a display face, a capture face, acoupling face, or a utility face) or an orientation of a second face(e.g., a display face, a capture face, a coupling face, or a utilityface) can be defined relative to an axis of symmetry for the firsttertiary structure or an axis of symmetry for the second tertiarystructure. In some configurations, an orientation of a first face may bethe same as an orientation of a second face (e.g., a face that hasdisplay and capture utility). An orientation of a first face may bedetermined with respect to an orientation of a second face based upon anangular offset between a first vector that is normal to a plane definingan average spatial location of the first face and a second vector thatis normal to a plane defining an average spatial location of the secondface. In other configurations, an orientation of a first face may beoffset from an orientation of a second face by at least about 90°. Inother configurations, an orientation of a first face may be offset froman orientation of a second face by about 180°. A nucleic acidnanostructure may comprise a first face and a second face with anangular offset of at least about 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°,90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, 170°, 180°, 190°, 200°,210°, 220°, 230°, 240°, 250°, 260°, 270°, 280°, 290°, 300°, 310°, 320°,330°, 340°, 350°, or more than 350°. Alternatively or additionally, anucleic acid nanostructure may comprise a first face and a second facewith an angular offset of no more than about 360°, 350°, 340°, 330°,320°, 310°, 300°, 290°, 280°, 270°, 260°, 250°, 240°, 230°, 220°, 210°,200°, 190°, 180°, 170°, 160°, 150°, 140°, 130°, 120°, 110°, 100°, 90°,80°, 70°, 60°, 50°, 40°, 30°, 20°, 10°, or less than 10°.

A nucleic acid nanostructure, such as a SNAP, may comprise a pluralityof tertiary or quaternary structures that at least partially surroundsor substantially encloses an internal volume region. A nucleic acidnanostructure may have a three-dimensional structure such as a pyramid,shell, cylinder, disk, sphere, cuboid (e.g., square cube or rectangularcuboid), or block, that comprises an internal volume region. An internalvolume region may be a three-dimensional volume within a nucleic acidnanostructure that is large enough to accommodate an analyte or othermolecule set forth herein. A nucleic acid nanostructure may beconfigured to comprise an internal volume region, where the internalvolume region comprises a utility face, such as a display face or acapture face. A utility moiety may be displayed within the internalvolume region. For example, a display moiety may be displayed within aninternal volume region of a SNAP such that an analyte is at leastpartially coupled within the internal volume region. In another example,a capture moiety may be displayed within an internal volume region of aSNAP such that a complementary moiety of a surface must at leastpartially enter the internal volume region to couple with the capturemoiety (see FIGS. 38A and 38B).

In some configurations, an internal volume region may be created in anucleic acid nanostructure (e.g., a SNAP) to control the interactionsbetween the nucleic acid nanostructure and other entities. An internalvolume region may comprise one or more moieties that alter the chemicalproperties (e.g., hydrophobicity, hydrophilicity, reactivity, polarity,solubility, etc.) of the internal volume region to differ from thechemical properties of the surrounding nucleic acid nanostructure. FIG.39A depicts a SNAP 3910 comprising an internal volume region 3920containing a capture moiety comprising a reactive group 3925 and aplurality of hydrophobic molecules 3928 surrounding the reactive group3925. The SNAP may be contacted with a surface 3930 comprising aplurality of hydrophilic groups 3932 terminated with complementaryreactive groups 3935 and a plurality of hydrophobic groups 3938terminated with complementary reactive groups 3935. As shown in FIG.39B, the hydrophobic property of the internal volume region 3920 mayincrease the likelihood that the SNAP 3910 will deposit and couple tothe surface 3930 at a region comprising the plurality of hydrophobicgroups 3938.

In some configurations, an internal volume region may be created in anucleic acid nanostructure (e.g., a SNAP) to control the interactions inwhich a moiety within the internal volume region may participate. Theorientation of a moiety within the internal volume region may becontrolled to increase, decrease, or otherwise control the orientationwith which an interaction may occur. A moiety may be displayed within aninternal volume region in a manner that limits or controls the size ofentities that may interact with the moiety. FIG. 38A depicts a SNAP 3810comprising an internal volume region 3820 containing a coupledmultivalent binding moiety (e.g., streptavidin, avidin) 3830. Thecoupled multivalent binding moiety 3830 is oriented within the internalvolume region 3820 such that only one binding site 3835 is available toparticipate in a binding interaction with an entity 3840 comprising acomplementary binding group (e.g., biotin) 3845 that is configured tocouple to the binding site 3835. As shown in FIG. 38B, the coupledmultivalent binding moiety 3830 has been made substantially monovalentdue to its orientation within the internal volume region 3820, therebyforming only one binding interaction with an entity 3840.

A nucleic acid nanostructure may comprise a first tertiary structuredomain and a second tertiary structure domain that are oriented withrespect to each other by one or more nucleic acid strands that formlinking strands (e.g., staple oligonucleotides) between the firsttertiary structure domain and the second tertiary structure domain. Alinking strand may comprise a single-stranded, double-stranded,partially double-stranded or multi-stranded nucleic acid. In someconfigurations, a nucleic acid nanostructure may comprise a firstoligonucleotide with a first nucleic acid sequence and a second nucleicacid sequence that hybridize to complementary sequences of a secondoligonucleotide to form a first tertiary structure domain and a secondtertiary structure domain, in which the first nucleic acid sequence andthe second nucleic acid sequence of the first oligonucleotide areseparated by a linking nucleic acid sequence that comprises asingle-stranded linking strand between the first tertiary structuredomain and the second tertiary structure domain. For example, the firstoligonucleotide can be a staple that hybridizes to a scaffold nucleicacid to form the first tertiary structure domain and the second tertiarystructure domain in a nucleic acid origami structure.

A nucleic acid nanostructure may comprise a first tertiary structuredomain and a second tertiary structure domain, in which a relativeangular orientation or spatial separation of the two domains iscontrolled by one or more linking strands. Angular orientation and/orspatial separation of a first tertiary structure domain and a secondtertiary structure domain may be tunable based upon the spatiallocations of nucleotides within the helical structure of the domains.Each complete revolution of a double-stranded nucleic acid helixtypically contains 10 to 11 nucleotide base pairs. Accordingly, theinitial angle of projection of a linking strand may be tuned by thenucleotide position within a helical structure. Tunability of structureof a nucleic acid nanostructure can also be obtained by varying a lengthof a linking strand and varying a separation distance betweenconsecutive linking strands. FIGS. 49A-49E depict aspects of controllingorientation of tertiary structures in a nucleic acid nanostructure. FIG.49A depicts a top-down view of a portion of a nucleic acid nanostructurecomprising a first oligonucleotide 4910 (e.g., a scaffold strand) and asecond oligonucleotide 4920 (e.g., a staple oligonucleotide), in whichthe second oligonucleotide 4920 hybridizes to the first oligonucleotide4910 to form a first tertiary structure domain 4930 and a secondtertiary structure domain 4932 that are connected by a linking strandcomprising a single-stranded nucleic acid sequence of the secondoligonucleotide 4920. FIGS. 49B-49C depict differences in initialorientation of the linking strand, as determined by nucleotide positionwithin a revolution of a helical structure, of the secondoligonucleotide 4920 as seen relative to the helical axes of the firsttertiary structure domain 4930 and the second tertiary structure domain4932. FIG. 49B depicts a configuration in which the initial orientationof linking strands is not coplanar, while FIG. 49C depicts aconfiguration in which the initial orientation of linking strands iscoplanar. Further, for a fixed length of a linking strand, thedifference in initial orientation of the linking strand may affect theseparation distance or amount of variation in separation distancebetween two neighboring tertiary structure domains, for example, asshown in FIGS. 49B and 49C. FIGS. 49D-49E illustrate possible relativepositions of the tertiary structure domains based upon the linkingstrand orientations, as shown in FIGS. 49B-49C, respectively. FIG. 49Ddepicts a skewed orientation between the first tertiary structure domain4930 and the secondary tertiary structure domain 4932, while FIG. 49Edepicts a coplanar orientation between the first tertiary structuredomain 4930 and the second tertiary structure domain 4932, with eachorientation of the two tertiary structure domains arising from thepositioning of the nucleotide at which the second oligonucleotide 4920transitions from a component of a double-stranded nucleic acid to asingle-stranded nucleic acid of the linking strand.

Location of linking strands may affect the conformation of a firsttertiary structure domain relative to a second tertiary structure domainin a nucleic acid nanostructure. For example, to configure a firsttertiary structure domain and a second tertiary structure domain in asubstantially coplanar orientation (i.e., a minimal angular offsetbetween the two tertiary structure domains), consecutive linking strandsmay be placed at about an odd number of helical half revolutions apart(e.g., about 1, 3, 5, 7, 9, etc. half turns or about 6, 16, 27, 37, 48,etc. nucleotides apart). Alternatively, to configure a first tertiarystructure domain and a second tertiary structure domain in a skewedorientation (i.e., a measurable angular offset between the two tertiarystructures), consecutive linking strands may be placed at positionsother than helical half revolutions, or may be placed at random orvarying positions including helical half revolutions and positions otherthan helical half-revolutions. For example, consecutive linking strandsmay be placed at about an even number of helical half revolutions apart(e.g., about 2, 4, 6, 8, 10, etc. half turns or about 11, 21, 31, 41,52, etc. nucleotides apart) or fractional numbers of helical halfrevolutions other than half revolutions (e.g., ¾ revolution, 1¾revolutions, 2¼ revolutions, etc.). In some configurations, it may bepreferable to produce a nucleic acid nanostructure that comprises asubstantially planar structure, in which the planar structure comprisesa plurality of coplanar tertiary structures. For example, a nucleic acidnanostructure may comprise a capture face that is substantially planarto increase an electrostatic interaction between the capture face and aplanar surface of a solid support. In other configurations, it may bepreferable to produce a nucleic acid nanostructure that comprises anon-planar structure comprising a plurality of tertiary structures, suchas a curved surface or a corrugated surface. For example, a nucleic acidnanostructure may comprise a capture face that comprises a corrugatedtexture to increase an electrostatic interaction between the captureface and a rough surface of a solid support.

A nucleic acid nanostructure may comprise one or more characteristics orconfigurations that deviate from characteristics or configurations ofnaturally-occurring nucleic acids. A nucleic acid nanostructure, as setforth herein, may comprise one or more non-natural nucleic acidstructures that increase the tunability of the nanostructure for one ormore purposes, such as the coupling and/or display of analytes, and thecoupling of the nanostructure to a solid support or a surface thereof. Anucleic acid nanostructure may be characterized by presence of one ormore non-natural nucleic acid structures, including but not limited to:i) a larger number of oligonucleotides hybridized to a given nucleicacid strand compared to the number of oligonucleotides hybridized to anatural nucleic acid strand of the same length and sequence, ii)increased volumetric and/or areal density of nucleotide packing within ananostructure or a component structure thereof compared to a naturalnucleic acid having the same or similar sequence content, iii) increasedsharpness of bending of a nucleic acid strand relative to anaturally-occurring nucleic acid having the same sequence or length, iv)decreased separation distance between non-contiguous regions of anucleic acid strand within a nanostructure compared to anaturally-occurring nucleic acid having the same sequence or length, v)low degree of sequence complementarity within a nanostructure relativeto the degree of sequence complementarity in a naturally-occurringnucleic acid that occupies a similar volume in solution, vi) greatermechanical rigidity of a nucleic acid strand in a nanostructure comparedto the mechanical rigidity of a naturally-occurring nucleic acid havingthe same sequence or length, and vii) combinations thereof.

A nucleic acid nanostructure, as set forth herein, may comprise morecomplexed oligonucleotides or nucleic acid strands than is known tooccur in a natural nucleic acid system such as a natural nucleic acidsystem having the same mass as the nucleic acid nanostructure.Naturally-occurring nucleic acids are predominantly nucleic acid strands(e.g., chromosomal DNA, plasmid strands) with partial or completecomplementary strands. Naturally-occurring nucleic acids may bedistinguished by complete or nearly-complete complementarity ofhybridized nucleic acid strands. Naturally-occurring nucleic acids maybe further distinguished by a relative small number of nucleic acidstrands complexed simultaneously by hybridization between each nucleicacid strand within the nucleic acid complex. For example, anaturally-occurring Holliday junction structure will typically involvethe hybridization of four nucleic acid strands, with each strand of thejunction complex having a high degree of sequence complementarity to twoother strands of the complex. Naturally-occurring nucleic acids oftenrequire additional proteins to complex multiple nucleic acid strands(e.g., chromosomal kinetochores, 3 nucleic acid complex during genetranscription formed by RNA polymerase, the RNA strand, and the twocomplementary DNA strands, etc.). In contrast, a nucleic acidnanostructure, as set forth herein, may comprise a larger quantity ofcomplex nucleic acid oligonucleotides or nucleic acid strands than isknown to occur in a natural nucleic acid system. For example, a nucleicacid nanostructure may comprise at least 10, 25, 50, 100, 150, 200, ormore than 200 complexed oligonucleotides or nucleic acid strands, inwhich each oligonucleotide or nucleic acid strand is hybridized to atleast one other oligonucleotide or nucleic acid strand of the nucleicacid nanostructure. In some configurations, a nucleic acid nanostructuremay be further characterized by an absence of a non-nucleic acidstructural element (e.g., a polypeptide, a protein, a polymer, ananoparticle) that is configured to join a first oligonucleotide ornucleic acid strand to a second oligonucleotide or nucleic acid strand.

A nucleic acid nanostructure, as set forth herein, may compriseincreased volumetric and/or areal density of nucleotide packing within ananostructure or a component structure thereof relative to anaturally-occurring nucleic acid such as a naturally-occurring nucleicacid having the same mass, nucleotide sequence or sequence length as thenucleic acid nanostructure. Naturally-occurring nucleic acids typicallyachieve volumetric nucleotide density through helical coiling ofdouble-stranded nucleic acids and supercoiling of helical nucleic acidsinto compacted structures. However, to achieve packing ofdouble-stranded nucleic acids with strand curvatures that exceed thepersistence length of double-stranded nucleic acids, naturally-occurringnucleic acids are typically complexed with proteins (e.g., histones)that condense helical nucleic acids into supercoiled structures. Incontrast, a nucleic acid nanostructure may comprise a volumetric densityof nucleotides that exceeds a volumetric nucleotide density of anaturally-occurring nucleic acid. A nucleic acid nanostructure mayachieve a greater volumetric nucleotide density than anaturally-occurring nucleic acid through increased bending and/orcurvature of nucleic acid structures and/or closer proximity of helicalstructures within the nucleic acid nanostructure. In someconfigurations, a nucleic acid nanostructure may achieve a greatervolumetric nucleotide density than a naturally-occurring nucleic acid inthe absence of a non-nucleic acid structural element (e.g., apolypeptide, a protein, a polymer, a nanoparticle) that is configured tocondense a nucleic acid structure.

A nucleic acid nanostructure, as set forth herein, may compriseincreased sharpness of bending of a nucleic acid relative to sequencelength and/or degree of secondary structuring relative to anaturally-occurring nucleic acid such as a naturally-occurring nucleicacid having the same nucleotide sequence or mass as the nucleic acidnanostructure. Naturally-occurring double-stranded nucleic acids have alarge persistence length that makes it unlikely that any portion of thedouble-stranded nucleic acid can approach within, for example, about 10nanometers of any other portion in the absence of a structure-alteringgroup (e.g., a histone). Even if single-stranded nucleic acid is presentwithin a naturally-occurring nucleic acid, two portions of tertiarystructure are unlikely to approach within, for example, about 10nanometers of each other due to electrostatic repulsion by negativelycharged polynucleotide backbones. Moreover, in the absence of a unifyingelement (e.g., a histone, a linking nucleic acid), two tertiarystructures are unlikely to remain stably oriented in a closeconfiguration in a naturally-occurring nucleic acid. In contrast, anucleic acid nanostructure, as set forth herein, may comprise sharplybent nucleic acid structures that increase the proximity of helicalstructures through the segmentation of double-stranded nucleic acidswith sequences of single-stranded nucleic acids. Neighboring helicalstructures may be held in close proximity by linking nucleic acidstrands that spatially and/or temporally stabilize the proximity andorientation of the neighboring helical structures relative to eachother. A nucleic acid nanostructure, as set forth herein, may be furtherdistinguished from naturally-occurring nucleic acids due to a presenceof a stable (i.e., spatially and/or temporally invariant) bend in anucleic acid strand that comprises two segmented regions of helicalstructure, for example a bend of at least 90° to 180°), relative to alength of a segment of single-stranded nucleic acid (e.g., no more than50, 40, 30, 25, 20, 15, or 10 nucleotides) of the nucleic acid strandthat separates the two segmented regions of helical structure.Alternatively or additionally, a nucleic acid nanostructure, as setforth herein, may be further distinguished from naturally-occurringnucleic acids due to a presence of a stable (i.e., spatially and/ortemporally invariant) bend in a nucleic acid strand that comprises twosegmented regions of helical structure, for example a bend of at least90° to 180°), relative to a degree of secondary structuring of thenucleic acid nanostructure (e.g., comprising at least about 80%, 85%,90%, or 95% of base-paired nucleotides relative to total nucleotidecontent).

A nucleic acid nanostructure, as set forth herein, may comprisedecreased separation distance between neighboring nucleic acidstructures within a nanostructure relative to a naturally-occurringnucleic acid such as a naturally-occurring nucleic acid having the samemass, nucleotide sequence or sequence length as the nucleic acidnanostructure. Adjacent helical (e.g., tertiary) structures may be heldin a temporally and/or spatially stable configuration at a distance of,for example, less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 nanometers.The close proximity of adjacent helical structures in nucleic acidnanostructures are unlikely to occur due to structural strain introducedby electrostatic repulsion of adjacent polynucleotide chains. Nucleicacid nanostructures may be capable of achieving close spatialproximities of helical structures and sharp bending angles of nucleicacid strands due to a presence of one or more linking nucleic acidstrands that stabilize the nucleic acid structure.

A nucleic acid nanostructure, as set forth herein, may comprise a lowdegree of sequence complementarity relative to total amount of nucleicacid present relative to a naturally-occurring nucleic acid such as anaturally-occurring nucleic acid having the same mass or sequence lengthas the nucleic acid nanostructure. A naturally-occurring nucleic acidstrand will typically be hybridized to a complementary nucleic acidstrand with an identical sequence length. Aside from replication orproofreading errors, the co-hybridized strands can be expected to havenear complete sequence complementarity, leading to an almost fullyhybridized structure in a stable configuration. In contrast, a nucleicacid nanostructure, as set forth herein, may comprise a plurality ofsingle-stranded nucleic acids within the nanostructure. Thesingle-stranded nucleic acids within a nucleic acid nanostructure may becharacterized as spatially and/or temporally stable, in contrast tonaturally-occurring nucleic acids, in which single-stranded nucleicacids are often formed and unformed transiently throughout the structureof the nucleic acid due to various biological processes. A nucleic acidnanostructure, as set forth herein, may comprise a stable fraction ofsingle-stranded nucleic acid as measured by percentage of unpairednucleotides within a nanostructure. In some configurations, a nucleicacid nanostructure may comprise a compacted region of predominantlydouble-stranded nucleic acids and a pervious region of predominantlysingle-stranded nucleic acids. In particular configurations, a nucleicacid nanostructure may comprise a compacted region of predominantlydouble-stranded nucleic acids and a pervious region of predominantlysingle-stranded nucleic acids, in which the pervious region comprises alarger total quantity of nucleotides than the compacted region. Anucleic acid nanostructure may comprise a spatially and/or temporallystable fraction of single-stranded nucleic acids as measured by unpairednucleotides, such as at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, ormore than 60% single-stranded nucleic acids.

A nucleic acid nanostructure, as set forth herein, may comprise greatermechanical rigidity relative to amount of single-stranded nucleic acidwithin a nanostructure when compared to naturally-occurring nucleicacids such as a naturally-occurring nucleic acids having the same mass,nucleotide sequence or sequence length as the nucleic acidnanostructure. For example, a strand of single-stranded nucleic acidwithin a linear double-stranded nucleic acid would typically createdecreased rigidity within the double-stranded nucleic acid as evidencedby increased relative motion between ends of the nucleic acid. Increasedamount of single-stranded nucleic acid within a linear double-strandednucleic acid would be expected to further decrease the amount ofrigidity of the nucleic acid. In contrast, a nucleic acid nanostructure,as set forth herein may comprise greater rigidity on a spatial and/ortemporal basis relative to total single-stranded nucleic acid contentrelative to a naturally-occurring nucleic acid with a samesingle-stranded nucleic acid content. The increased rigidity may arisedue to linking strands that stabilize nucleic acid structures relativeto each other within the nucleic acid nanostructure.

Nucleic Acid Configurations: Described herein are nucleic acidnanostructures such as SNAPs. The nucleic acid nanostructures may beutilized for multiple purposes, including the display of molecules oranalytes at a surface or interface, such as a solid support or a phaseboundary. The described nucleic acid nanostructures, such as SNAPs, maycomprise various primary, secondary, tertiary, or quaternary structuresthat give rise to compacted nucleic acid particles with variousgeometries that add utility to the nanostructures. Any given nucleicacid nanostructure may serve one or more functions, including displayinga molecule or an analyte (a display SNAP), or performing othernanostructure-related utilities (a utility SNAP). A nucleic acidnanostructure, such as a utility SNAP, may perform such functions ascoupling a molecule or an analyte to a surface or interface (a captureSNAP), coupling a nucleic acid nanostructure to another nucleic acidnanostructure (a coupling SNAP), providing other structural utilities toa nucleic acid nanostructure or a complex thereof (a structural SNAP),or a combination thereof. In some configurations, a nucleic acidnanostructure may comprise a display SNAP, a utility SNAP, or acombination thereof. For example, a nucleic acid nanostructure (e.g., aSNAP) may be configured to couple to an analyte and a solid support,thereby making the nucleic acid nanostructure both a displaynanostructure and a utility nanostructure.

A nucleic acid nanostructure, such as a SNAP, may comprise a displayface that contains a display moiety. A display moiety may be configuredto couple an analyte by a suitable interaction, such as a covalent bond,a non-covalent interaction, an electrostatic interaction, or a magneticinteraction. A display moiety may comprise one or more functionalgroups, ligands, or other moieties that are configured to couple ananalyte. A display moiety may comprise a residue of a nucleic acid, ormay comprise a functional group, ligand, or moiety coupled thereto. Adisplay moiety may further comprise one or more secondary, tertiary, orquaternary structures that are positioned within a display face. Anucleic acid nanostructure, such as a SNAP, may comprise a capture facethat contains a capture moiety. The capture moiety may be configured tocouple to a surface by a suitable interaction, such as a covalent bond,a non-covalent interaction, an electrostatic interaction, or a magneticinteraction. A capture moiety may comprise one or more functionalgroups, ligands, or other moieties that are configured to couple to asurface. A capture moiety may further comprise one or more secondary,tertiary, or quaternary structures that are positioned within a captureface.

A display moiety may include two or more display tertiary structures ofa plurality of tertiary structures. A capture moiety may include two ormore capture tertiary structures of a plurality of tertiary structures.In some configurations, a display tertiary structure of the two or moredisplay tertiary structures may comprise a capture tertiary structure ofthe two or more capture tertiary structures. For example, in FIG. 2B,face T may comprise the display moiety and face B may comprise thecapture moiety, with the four tertiary structures belonging to bothmoieties. In other configurations, the two or more display tertiarystructures do not comprise any capture tertiary structure of the two ormore capture tertiary structures. For example, in FIG. 2D, the displaymoiety may comprise the two tertiary structures associated with face Dand the capture moiety may comprise the two tertiary structuresassociated with face B. In some configurations, the two or more capturetertiary structures do not comprise any display tertiary structure ofthe two or more display tertiary structures.

A nucleic acid nanostructure, such as a SNAP, may comprise a pluralityof nucleic acid strands, the strands being molecules that are separableone from another without breaking covalent bonds. For example, a SNAPmay comprise a nucleic acid molecule that forms a scaffold strand and aplurality of staple oligonucleotide molecules hybridized to the scaffoldstrand. In some configurations, a scaffold strand may comprise anoligonucleotide of a plurality of oligonucleotides, in which theoligonucleotide is coupled to a greater quantity of oligonucleotides ofthe plurality of oligonucleotides than any other oligonucleotide of theplurality of oligonucleotides. A scaffold strand may comprise a linear,branched, or circular polynucleotide. In some configurations, a nucleicacid nanostructure may comprise two or more scaffold strands, such asabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more scaffold strands, whereeach strand is optionally a molecule that is separable from the otherstrand(s) of the nucleic acid nanostructure. A nucleic acidnanostructure with two or more scaffold strands may comprise a firstscaffold strand that is linked to a second scaffold strand by one ormore oligonucleotides of the plurality of oligonucleotides that arehybridized to the first scaffold strand and the second scaffold strand.A first scaffold strand may be linked to a second scaffold strand by acertain number of the plurality of oligonucleotides, such as, forexample, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or more than 50%of oligonucleotides in the plurality of oligonucleotides. Alternativelyor additionally, a first scaffold strand may be linked to a secondscaffold strand by no more than about 50%, 49%, 48%, 47%, 46%, 45%, 44%,43%, 42%, 41% 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31% 30%, 29%,28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lessthan 1% of oligonucleotides in the plurality of oligonucleotides.

A nucleic acid scaffold may comprise a continuous strand of nucleicacids that, with or without complementary oligonucleotides, is acircular or joined strand (i.e., the scaffold strand having no 5′ or 3′termini). In some configurations, a scaffold strand is derived from anatural source, such as a viral genome or a bacterial plasmid. In otherconfigurations, a scaffold strand may be engineered, rationallydesigned, or synthetic, in whole or in part. A scaffold strand maycomprise one or more modified nucleotides. Modified nucleotides mayprovide conjugation sites for attaching additional components, such asaffinity reagents or detectable labels. A modified nucleotide may beutilized as a conjugation site for an additional component (e.g. bindingcomponent or label component) before, during, or after assembly of anucleic acid nanostructure, such as a SNAP. A modified nucleotide mayinclude a linking group or a reactive handle (e.g., a functional groupconfigured to perform a click reaction). In some configurations, anucleic acid scaffold may comprise a single strand of an M13 viralgenome. The size of a scaffold strand may vary depending upon thedesired size of a nucleic acid nanostructure. A scaffold strand maycomprise a length of at least about 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800,7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000, 9500,10000, or more than 10000 nucleotides. Alternatively or additionally, ascaffold strand may comprise a length of at most about 10000, 9500,9000, 8800, 8600, 8400, 8200, 7800, 7600, 7400, 7200, 7000, 6800, 6600,6400, 6200, 6000, 5800, 5600, 5400, 5200, 5000, 4500, 4000, 3500, 3000,2500, 3000, 2500, 2000, 1500, 1000 or less than 1000 nucleotides.

A nucleic acid nanostructure, such as a SNAP, may comprise a pluralityof staple oligonucleotides. A staple oligonucleotide may comprise anyoligonucleotide that is hybridized with, or configured to hybridizewith, a nucleic acid scaffold, other staples, or a combination thereof.A staple oligonucleotide may be modified to include additional chemicalentities, such as binding components, label components,chemically-reactive groups or handles, or other groups (e.g.,polyethylene glycol (PEG) moieties). A staple oligonucleotide maycomprise linear or circular nucleic acids. A staple oligonucleotide maycomprise one or more single-stranded regions, double-stranded regions,or combinations thereof. A staple oligonucleotide may be hybridizedwith, or configured to hybridize with, a scaffold strand or one or moreother staples, for example, via complementary base pair hybridization(e.g., Watson-Crick hybridization). A staple oligonucleotide may behybridized with other nucleic acids by complementary base pairhybridization or ligation. A staple oligonucleotide may be configured toact as a primer for a complementary nucleic acid strand and the primerstaple may be extended by an enzyme (e.g., a polymerase) to formlengthened regions of double-stranded nucleic acid, for example, using ascaffold, staple or other strand as a template. In some cases the primerneed not be hybridized to a template when extended. For example, aprimer can be extended by template-free addition of one or morenucleotides by a terminal transferase enzyme, by template-free additionof one or more oligonucleotides by a ligase enzyme or template-freeaddition of nucleotide(s) or oligonucleotide(s) by non-enzymaticchemical reaction. A staple oligonucleotide may include one or moremodified nucleotides. A modified nucleotide may include a linking groupor a reactive handle (e.g., a functional group configured to perform aclick-type reaction).

A staple oligonucleotide may be any length depending upon the design ofthe SNAP. A staple oligonucleotide may be designed by a softwarepackage, such as caDNAno², ATHENA, OR DAEDALUS. A staple oligonucleotidemay have a length of at least about 10, 25, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000,3500, 4000, 4500, 5000, or more than 5000 nucleotides. Alternatively oradditionally, a staple may have a length of no more than about 5000,4500, 4000, 3500, 3000, 2500, 2000, 1900, 1800, 1700, 1600, 1500, 1400,1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550,500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 25, 10, or less than 10nucleotides.

A staple may comprise a first nucleotide sequence and a secondnucleotide sequence, in which the first nucleotide sequence hybridizedto a first complementary sequence, and in which the second nucleotidesequence is hybridized to a second complementary sequence. In someconfigurations, a staple may comprise a first nucleotide sequence and asecond nucleotide sequence, in which the first nucleotide sequence ishybridized to a first complementary sequence, in which the secondnucleotide sequence is hybridized to a second complementary sequence,and in which the first nucleotide sequence is linked to the secondnucleotide sequence by a linking moiety (e.g., a linker as set forthherein, an intermediate single-stranded nucleotide sequence, anintermediate double-stranded nucleotide sequence, an intermediatenucleotide sequence that is not configured to couple to a complementarynucleotide sequence, etc.). In some configurations, a staple maycomprise a first nucleotide sequence and a second nucleotide sequence,in which the first nucleotide sequence is hybridized to a firstcomplementary sequence of a scaffold strand, and in which the secondnucleotide sequence hybridized to a second complementary sequence of thescaffold strand. In particular configurations, a first complementarysequence and a second complementary sequence of a scaffold strand may benon-consecutive, such that the two complementary sequence regions areseparated by a third region of the scaffold strand. A staple maycomprise a first nucleotide sequence and a second nucleotide sequence,in which the first nucleotide sequence is hybridized to a firstcomplementary sequence, and in which the second nucleotide sequence isnot hybridized to a second complementary sequence (e.g., a pendantmoiety). A first nucleotide sequence or a second nucleotide sequence ofa staple oligonucleotide may comprise a sequence length of at leastabout 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides.Alternatively or additionally, a first nucleotide sequence or a secondnucleotide sequence of a staple oligonucleotide may comprise a sequencelength of no more than about 30, 29, 28 27, 26, 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or lessthan 3 nucleotides. A sequence length of a nucleotide sequence of astaple oligonucleotide may be chosen to provide a hybridized nucleicacid containing the staple oligonucleotide a particular meltingtemperature, as set forth herein.

A staple oligonucleotide may include one or more modified nucleotides.Modified nucleotides may provide conjugation sites for attachingadditional components, such as binding components or label components. Amodified nucleotide may increase the stability of an oligonucleotide tochemical degradation, e.g., a locked nucleic acid (LNA). A modifiednucleotide may be utilized as a conjugation site for an additionalcomponent before, during, or after assembly of a nucleic acidnanostructure, such as a SNAP. A staple oligonucleotide may include atleast about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more than 100modified nucleotides. Alternatively or additionally, A stapleoligonucleotide may include no more than about 100, 75, 50, 45, 40, 35,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or less than 2 modified nucleotides.

A nucleic acid nanostructure, as set forth herein, may comprise aplurality of nucleic acids, in which each nucleic acid of the pluralityof nucleic acids is hybridized to one or more other nucleic acid of theplurality of nucleic acids. In some configurations, a nucleic acidnanostructure may comprise at least 5 nucleic acids, in which eachnucleic acid of the at least 5 nucleic acids is coupled to one or moreother nucleic acids of the at least 5 nucleic acids. A plurality ofnucleic acids of a nucleic acid nanostructure may comprise a scaffoldstrand, in which the scaffold strand is characterized by one or morecharacteristics of: i) comprising a longest nucleotide sequence of theplurality of nucleic acids, and ii) being configured to hybridize with agreater quantity of other nucleic acids of the plurality of nucleicacids. A plurality of nucleic acids of a nucleic acid nanostructure mayfurther comprise one or more staple oligonucleotides, in which a stapleoligonucleotide is characterized by one or more characteristics of: i)comprising two or more non-consecutive nucleotide sequences that areconfigured to hybridize to one or more other nucleic acids (e.g., one ormore regions of a scaffold strand, a scaffold strand and a second stapleoligonucleotide, a second staple oligonucleotide and a third stapleoligonucleotide, etc.), ii) comprising two or more non-consecutivenucleotide sequences that are configured to form two or more secondaryand/or tertiary structures when hybridized with one or more othernucleic acids, ii) comprising one or more nucleotide sequences that arenot configured to hybridize to other nucleic acids, and iii) comprisingone or more nucleotide sequences that are configured to constrain aposition, orientation, and/or motion of a first secondary and/ortertiary nucleic acid structure relative to a second secondary and/ortertiary nucleic acid structure.

FIG. 51 illustrates a schematic of a nucleic acid nanostructurecomprising a scaffold strand 5101 and a plurality of stapleoligonucleotides, in which the staple oligonucleotides have a variety ofstructural and/or functional roles. The nucleic acid nanostructurecomprises a plurality of structural staple oligonucleotides that eachhave one or more properties of: i) binding with the scaffold strand 5101to form one or more tertiary structures, and ii) forming linkingsingle-stranded nucleic acids that position and orient two or moretertiary structures of the nucleic acid nanostructure with respect toeach other. Structural staple oligonucleotides include: 1) nucleic acid5104, which binds to the scaffold strand 5101 to form a region oftertiary structure, 2) nucleic acid 5107, which binds to the scaffoldstrand 5101 at two nucleotide sequences to form a substantially 180°bend in the nucleic acid nanostructure and links the two tertiarystructures formed by the binding of the nucleic acid 5107 to thescaffold strand 5101 by a linking strand comprising a single-strandednucleotide sequence of nucleic acid 5107, 3) nucleic acid 5108, whichbinds to the scaffold strand 5101 at three non-consecutive nucleotidesequences to form at least 3 tertiary structures and 2 substantially180° bends in the nucleic acid nanostructure, and 4) nucleic acids 5109,which each comprise a first sequence that is complementary to thescaffold strand 5101 and a second sequence that is complementary to theother nucleic acid 5109 to form a 3 tertiary structures and 1substantially 180° bend in the nucleic acid nanostructure. A nucleicacid nanostructure may also comprise a non-nucleic acid structuralelement 5110, such as a nucleic-acid binding protein (e.g., a histone)or a nanoparticle, in which the non-nucleic acid structural element 5110forms or stabilizes a portion of the two-dimensional and/orthree-dimensional structure of the nucleic acid nanostructure. Thenucleic acid nanostructure further comprises a plurality of functionalstaple oligonucleotides that each have one or more properties of: i)binding with the scaffold strand 5101 to form one or more tertiarystructures, and ii) modifying the nucleic acid nanostructure to provideadditional chemical and/or physical properties to the nucleic acidnanostructure. Functional staple oligonucleotides include: 1) nucleicacid 5102, which binds to the scaffold strand 5101 to form a tertiarystructure and comprises a moiety 5103 (e.g., a terminal ligand, anon-terminal ligand, a terminal functional group, a non-terminalfunctional group, a modified nucleotide, a non-nucleic acid polymer,etc.), 2) nucleic acid 5105, which binds to the scaffold strand 5101 toform a tertiary structure and comprises a detectable label 5106 (e.g., afluorophore, a nucleic acid barcode, a peptide barcode, etc.), 3)pendant nucleic acid 5111, which binds to the scaffold strand 5101 toform a tertiary structure and comprises an uncoupled terminal residue ornucleotide sequence, 4) pendant nucleic acid 5112, which comprises twouncoupled terminal residues or nucleotide sequences and an intermediatenucleotide sequence that binds to the scaffold strand 5101 to form atertiary structure, and 5) pendant nucleic acid 5113, which comprise twoterminal nucleotide sequences that bind to the scaffold strand 5101 toform tertiary structures and an intermediate single-stranded nucleotidesequence that is pendant from the nucleic acid nanostructure (includingone or more coupled oligonucleotides 5114 that provide tertiarystructuring to the pendant portion of nucleic acid 5113.

A nucleic acid nanostructure, such as a SNAP can include a nucleic acidorigami. Accordingly, a nucleic acid nanostructure can include one ormore nucleic acids having tertiary or quaternary structures such asspheres, cages, tubules, boxes, tiles, blocks, trees, pyramids, wheels,combinations thereof, and any other possible structure. Examples of suchstructures formed with DNA origami are set forth in Zhao et al. NanoLett. 11, 2997-3002 (2011), which is incorporated herein by reference.In some configurations, a nucleic acid nanostructure, such as a SNAP,may comprise a scaffold strand and a plurality of stapleoligonucleotides, where the scaffold strand is a single, continuousstrand of nucleic acid, and the staple oligonucleotides are configuredto bind, in whole or in part, with the scaffold strand. Examples of DNAorigami structures formed using a continuous scaffold strand and severalstaple strands are set forth in Rothemund Nature 440:297-302 (2006) andU.S. Pat. Nos. 8,501,923 and 9,340,416, each of which is incorporatedherein by reference. A nucleic acid nanostructure comprising one or morenucleic acids (e.g., as found in origami or nanoball structures) maycomprise regions of single-stranded nucleic acid, regions ofdouble-stranded nucleic acid, or combinations thereof. In someconfigurations, a nucleic acid nanostructure may comprise a nucleic acidorigami and a nucleic acid structure other than a nucleic acid origami.For example, a nucleic acid origami may be coupled to one or moresingle-stranded nucleic acids, in which the one or more single-strandednucleic acids do not form any secondary and/or tertiary structures. Inan advantageous configuration, a nucleic acid origami may comprise atile structure. A tile structure of a nucleic acid origami may refer toa structure with an average thickness that is substantially smaller thana characteristic dimension (e.g., side length, side width, maximumdiameter, average diameter, etc.). For example, a tile structure of anucleic acid origami may have an aspect ratio between a characteristicdimension and an average thickness of at least about 2:1, 3:1, 4:1, 5:1,10:1, 20:1, or more than 20:1. Alternatively or additionally, a tilestructure may have an aspect ratio between a characteristic dimensionand an average thickness of no more than about 20:1, 10:1, 5:1, 4:1,3:1, 2:1, or less than 2:1. A tile structure may have a shape, such as asubstantially rectangular tile, a substantially square tile, asubstantially triangular tile, a substantially circular tile, asubstantially oval tile, or a substantially polygonal tile. A tile maycomprise one or more faces that are substantially planar. A tile maycomprise one or more faces that are substantially non-planar (e.g.,curved, corrugated, etc.).

A nucleic acid nanostructure, such as a SNAP, may comprise two or moreutility faces that are formed by the scaffold strand hybridizing to theplurality of staple oligonucleotides. The hybridizing of the pluralityof staple oligonucleotides to the scaffold strand may form a pluralityof tertiary nucleic acid structures in a nucleic acid nanostructure. Insome configurations, a plurality of tertiary structures may comprise afirst tertiary structure belonging to a first utility face (e.g., adisplay face) and a secondary tertiary structure belonging to a secondutility face (e.g., a capture face). Two tertiary structures in anucleic acid nanostructure (e.g., a SNAP) may be oriented with respectto each other relative to an axis or plane of symmetry. Two tertiarystructures in a nucleic acid nanostructure may be oriented with respectto each other relative to an axis or plane of symmetry of one or both ofthe tertiary structures, such as the coaxial axis of symmetry for anucleic acid double helix. In some configurations with a first andsecond tertiary structure belonging to differing utility faces, the axisof symmetry of the first tertiary structure and the axis of symmetry ofthe second tertiary structure are coplanar. For configurations in whicha first and second tertiary structure belong to differing utility faces,the axis of symmetry of the first tertiary structure and the axis ofsymmetry of the second tertiary structure can be non-coplanar. In someconfigurations in which a first and second tertiary structure belong todiffering utility faces, the axis of symmetry of the first tertiarystructure and the axis of symmetry of the second tertiary structure canbe intersecting. In some configurations in which a first and secondtertiary structure belong to differing utility faces, the axis ofsymmetry of the first tertiary structure and the axis of symmetry of thesecond tertiary structure can be non-intersecting. A symmetrycharacteristic of a nucleic acid nanostructure (e.g., a SNAP) may bedetermined with respect to an average dimension, shape, or configurationof the nucleic acid nanostructure. Slight variations in positioning offeatures, for example, due to the helical structure and tertiarystructures of a nucleic acid nanostructure or temporal variations due toenvironmental conditions (e.g., Brownian motion, fluidic shear,electromagnetic forces, etc.), may cause small differences between twoopposed sides of a nucleic acid nanostructure that is designed to have asymmetrical structure. A nucleic acid nanostructure may be consideredsymmetric if two symmetric features lie within about 10% of the expectedposition with respect to an axis or plane of symmetry.

A nucleic acid nanostructure composition (e.g., a SNAP composition) mayfurther comprise a molecule or an analyte. Optionally, the molecule oranalyte is a non-nucleic acid molecule or analyte, respectively. In someconfigurations, a display moiety of a nucleic acid nanostructure may becoupled to the molecule or analyte. For example, a plurality of SNAPsmay be deposited on an array after each SNAP of the plurality of SNAPshas been coupled to the molecule or analyte. In other configurations, adisplay moiety of a nucleic acid nanostructure need not be coupled to amolecule or an analyte. For example, a plurality of SNAPs may bedeposited on an array before each SNAP of the plurality of SNAPs hasbeen coupled to a molecule or an analyte. In some configurations, amolecule or an analyte may comprise a biomolecule selected from thegroup consisting of polypeptide, polysaccharide, nucleic acid, lipid,metabolite, enzyme cofactor, and a combination thereof. In someconfigurations, a molecule or an analyte may comprise a non-biologicalparticle selected from the group consisting of polymer, metal, metaloxide, ceramic, semiconductor, mineral, and a combination thereof.

A nucleic acid nanostructure composition (e.g., a SNAP composition) maycomprise a linker that is configured to couple an entity (e.g., a SNAP,an analyte, a coupling surface, etc.) to a moiety (e.g., asurface-interacting moiety, a display moiety, a capture moiety, asurface-linked moiety, etc.). A linker may have a size of at least about100 Da, 500 Da, 1 kDa, 5 kDa, 10 kDa, 20 kDa, 25 kDa, 50 kDa, 100 kDa,250 kDa, 500 kDa, or more than 500 kDa. Alternatively or additionally, alinker may have a size of no more than about 500 kDa, 250 kDa, 100 kDa,50 kDa, 25 kDa, 20 kDa, 10 kDa, 5 kDa, 1 kDa, 500 Da, 100 Da, or lessthan about 100 Da. A linker may comprise a chemical physical property(e.g., hydrophobicity, hydrophilicity, polarity, steric size, netelectrical charge, etc.) that mediates an interaction between an entityand a moiety that are joined by the linker. For example, a SNAP maycomprise a rigid linker that separates an analyte of interest from asurface by a separation distance and/or prevents contact between theanalyte of interest and a face of the SNAP.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a functionalnucleic acid. A functional nucleic acid may bring an additional utilityto a nucleic acid nanostructure. A functional nucleic acid may comprisea nucleic acid barcode that may provide a tagging or informationencoding function, for example, in the form of an identifying sequencefor an analyte that is colocalized with the functional nucleic acid. Asshown in FIGS. 10A-10D, the utility moiety 1040 may comprise a nucleicacid barcode sequence that may be transcribed onto a molecule thatinteracts with the analyte 1020, or vice versa. A barcode sequencecontained on a utility moiety 1040 or an interacting molecule may besequenced to determine a characteristic or prior use of analyte 1020,such as any interactions that may have occurred with the analyte 1020. Afunctional nucleic acid may comprise a retaining moiety, in which theretaining moiety comprises a hybridizing nucleic acid sequence that isconfigured to form a short-term or weak interaction that temporarilyco-locates an interacting molecule in the vicinity of the analyte toincrease the likelihood of an interaction being observed or to decreasethe rate at which the interacting molecule dissociates from the analyte.A hybridizing nucleic acid sequence may comprise a short region ofcomplementarity with another oligonucleotide (e.g., less than about 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides), anucleic acid sequence with imperfect complementarity to another nucleicacid, a toehold sequence, or any other configuration that promotes aneasily reversible nucleic acid hybridization interaction. A functionalnucleic acid may comprise a nucleic acid sequence that is configured tobind a labeled nucleic acid (e.g., a fluorescently-labeledoligonucleotide) for a purpose such as detecting a spatial address of anucleic acid nanostructure (e.g., on a site of a solid support).

In another aspect, provided herein is a method of forming a multiplexarray of analytes, comprising: a) contacting an array comprising aplurality of sites with a first plurality of nucleic acidnanostructures, as set forth herein, in which each nucleic acidnanostructure of the first plurality of nucleic acid nanostructures iscoupled to an analyte of interest of a first plurality of analytes ofinterest, b) contacting the array comprising the plurality of sites witha second plurality of nucleic acid nanostructures, as set forth herein,in which each nucleic acid nanostructure of the second plurality ofnucleic acid nanostructures is coupled to an analyte of interest of asecond plurality of analytes of interest, c) depositing the firstplurality of nucleic acid nanostructures at a first subset of sites ofthe plurality of sites, and d) depositing the second plurality ofnucleic acid nanostructures at a second subset of sites of the pluralityof sites, in which the first subset of sites and the second subset ofsites comprise a random spatial distribution. In some configurations,each nucleic acid nanostructure of the first plurality of nucleic acidnanostructures may comprise a first functional nucleic acid, in whichthe first functional nucleic acid comprises a first nucleotide sequence,in which each nucleic acid nanostructure of the second plurality ofnucleic acid nanostructures may comprise a second functional nucleicacid, in which the second functional nucleic acid comprises a secondnucleotide sequence, and in which the first nucleotide sequence differsfrom the second nucleotide sequence. In some configurations, a method offorming a multiplex array may comprise simultaneously contacting thearray with the first plurality of nucleic acid nanostructure and thesecond plurality of nucleic acid nanostructures. For example, an arraymay be contacted with a fluidic medium containing a mixture of the firstplurality of nucleic acid nanostructures and the second plurality ofnucleic acid nanostructures. In other configurations, a method offorming a multiplex array may comprise sequentially contacting the arraywith the first plurality of nucleic acid nanostructure and the secondplurality of nucleic acid nanostructures. In some configurations, amethod of forming a multiplex array may comprise simultaneouslydepositing on the array the first plurality of nucleic acidnanostructure and the second plurality of nucleic acid nanostructures.For example, an array may be contacted with a fluidic medium containinga mixture of the first plurality of nucleic acid nanostructures and thesecond plurality of nucleic acid nanostructures, then contacted with asecond fluidic medium that facilitates the deposition of the nucleicacid nanostructures onto sites of the array. In other configurations, amethod of forming a multiplex array may comprise sequentially depositingon the array the first plurality of nucleic acid nanostructure and thesecond plurality of nucleic acid nanostructures.

A method of forming a multiplex array of analytes may further comprise astep of contacting the array with a first plurality of detectablenucleic acids, in which each first detectable nucleic acid of the firstplurality of detectable nucleic acids comprises a first complementarynucleotide sequence and a detectable label, in which the firstcomplementary nucleotide sequence is complementary to a first nucleotidesequence of a first functional nucleic acid of a nucleic acidnanostructure of the first plurality of nucleic acid nanostructures.After contacting the array with the first plurality of detectablenucleic acids, a method of forming a multiplex array of analytes mayfurther comprise coupling a first detectable nucleic acid to each firstfunctional nucleic acid. After coupling the first detectable nucleicacid to each first functional nucleic acid, the method may furthercomprise a step of detecting each address of the array comprising thefirst detectable nucleic acid, as set forth herein. After coupling thefirst detectable nucleic acid to each first functional nucleic acid, themethod may further comprise a step of removing the first detectablenucleic acid from the first functional nucleic acid. In someconfigurations, removing the first detectable nucleic acid from thefirst functional nucleic acid may comprise heating a nucleic acidnanostructure of the first plurality of nucleic acid nanostructures toat least a melting temperature of the first functional nucleic acid,thereby uncoupling the first detectable nucleic acid from the firstfunctional nucleic acid. In other configurations, removing a firstdetectable nucleic acid from the first functional nucleic acid maycomprise contacting a solid support with a fluidic medium that isconfigured to separate the first detectable nucleic acid from the firstfunctional nucleic acid (e.g., a denaturant, a chaotrope, etc.),optionally in the presence of heating.

A method of forming a multiplex array of analytes may comprisecontacting the array with two or more pluralities of detectable nucleicacids. For example, a method exemplified above, may further comprise astep of contacting the array with a second plurality of detectablenucleic acids, in which each second detectable nucleic acid of thesecond plurality of detectable nucleic acids comprises a secondcomplementary nucleotide sequence and a detectable label, in which thesecond complementary nucleotide sequence is complementary to a secondnucleotide sequence of a second functional nucleic acid of a nucleicacid nanostructure of the second plurality of nucleic acidnanostructures. After contacting the array with the second plurality ofdetectable nucleic acids, a method of forming a multiplex array ofanalytes may further comprise coupling a second detectable nucleic acidto each second functional nucleic acid. After coupling the seconddetectable nucleic acid to each second functional nucleic acid, themethod may further comprise a step of detecting each address of thearray comprising the second detectable nucleic acid, as set forthherein. After coupling the second detectable nucleic acid to each secondfunctional nucleic acid, the method may further comprise a step ofremoving the second detectable nucleic acid from the second functionalnucleic acid. In some configurations, removing the second detectablenucleic acid from the second functional nucleic acid may compriseheating a nucleic acid nanostructure of the second plurality of nucleicacid nanostructures to at least a melting temperature of the secondfunctional nucleic acid, thereby uncoupling the second detectablenucleic acid from the second functional nucleic acid. In otherconfigurations, removing a second detectable nucleic acid from thesecond functional nucleic acid may comprise contacting a solid supportwith a fluidic medium that is configured to separate the seconddetectable nucleic acid from the second functional nucleic acid (e.g., adenaturant, a chaotrope, etc.), optionally in the presence of heating.

FIGS. 50A-50F depict a method of utilizing a functional nucleic acid forforming a multiplexed array of analytes of interest. FIG. 50Aillustrates an array comprising a solid support 5000 comprising aplurality of sites 5001, with each site coupled to a SNAP 5010. Thesolid support 5000 is contacted with a plurality of SNAPs 5010. A firstsubset of the plurality of SNAPs 5010 comprise a SNAP 5010 coupled to afirst analyte of interest 5020 (e.g., polypeptides from a first sample),in which each SNAP 5010 of the first subset comprises a first functionalnucleic acid 5030 containing a nucleotide sequence of CGT. A secondsubset of the plurality of SNAPs comprise a SNAP 5010 coupled to asecond analyte of interest 5025 (e.g., polypeptides from a secondsample), in which each SNAP 5010 of the second subset comprises a secondfunctional nucleic acid 5035 containing a nucleotide sequence of CCA.FIG. 50B illustrates a multiplexed array formed by deposition of theplurality of SNAPs 5010 at the plurality of sites 5001 on the solidsupport 5010. The first subset of SNAPs 5010 and the second subset ofSNAPs 5010 comprise a random spatial distribution at the plurality ofsites 5001, in which the addresses of first analytes of interest 5020and second analytes of interest 5025 on the array are not initiallyknown after deposition. FIG. 50C depicts contacting the solid support5000 with a first plurality of detectable nucleic acids, in which eachdetectable nucleic acid comprises a detectable label 5045 and acomplementary nucleic acid 5040 with a nucleotide sequence of GCA. FIG.50D depicts the multiplexed array of SNAPs 5010, in which the firstsubset of SNAPs 5010 have coupled a detectable nucleic acid of the firstplurality of detectable nucleic acids by base-pair bonding between thefirst functional nucleic acids 5030 and the complementary nucleic acids5040. Each site 5001 comprising a first analyte of interest 5020 may bedetectable at single-analyte resolution by detection of the detectablelabel 5045 at addresses on the array. FIG. 50E depicts contacting thesolid support 5000 with a second plurality of detectable nucleic acids,in which each detectable nucleic acid comprises a detectable label 5046and a complementary nucleic acid 5041 with a nucleotide sequence of GGT.FIG. 50F depicts the multiplexed array of SNAPs 5010, in which thesecond subset of SNAPs 5010 have coupled a detectable nucleic acid ofthe second plurality of detectable nucleic acids by base-pair bondingbetween the second functional nucleic acids 5035 and the complementarynucleic acids 5041. Each site 5001 comprising a first analyte ofinterest 5025 may be detectable at single-analyte resolution bydetection of the detectable label 5046 at addresses on the array. Insome configurations, the addresses of the first analytes of interest5020 and the second analytes of interest 5025 can be simultaneouslydetected, for example by the use of detectable labels 5045 and 5046(e.g., fluorophores) with differing detection characteristics (e.g.,excitation wavelength, emission wavelength).

A functional nucleic acid, as set forth herein, may comprise anucleotide sequence that is configured to hybridize with a complementarynucleotide sequence of a coupled moiety (e.g., a detectable label, anucleic acid barcode, a retaining moiety, etc.). A functional nucleicacid may comprise a nucleotide sequence that is configured to form adouble-stranded nucleic acid with a complementary nucleic acid, in whichthe double-stranded nucleic acid is disruptable by melting of thedouble-stranded nucleic acid. A double-stranded functional nucleic acidmay have a melting temperature of at least about 50° C., 55° C., 60° C.,61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C.,70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C.,79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C.,88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C.,97° C., 98° C., 99° C., or more than 99° C. Alternatively oradditionally, a double-stranded functional nucleic acid may have amelting temperature of no more than about 99° C., 98° C., 97° C., 96°C., 95° C., 94° C., 93° C., 92° C., 91° C., 90° C., 89° C., 88° C., 87°C., 86° C., 85° C., 84° C., 83° C., 82° C., 81° C., 80° C., 79° C., 78°C., 77° C., 76° C., 75° C., 74° C., 73° C., 72° C., 71° C., 70° C., 69°C., 68° C., 67° C., 66° C., 65° C., 64° C., 63° C., 62° C., 61° C., 60°C., 55° C., 50° C., or less than 50° C. In some configurations, amelting temperature of a double-stranded functional nucleic acid of anucleic acid nanostructure may be designed to be lower than a meltingtemperature of some or all other double-stranded nucleic acids of thenucleic acid nanostructure. In a particular configuration, a meltingtemperature of a double-stranded functional nucleic acid of a nucleicacid nanostructure may be designed to be lower than a meltingtemperature of at least 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%of some or all of the double-stranded nucleic acids of the nucleic acidnanostructure. For example, a functional nucleic acid may be separatedfrom a complementary nucleic acid at a melting temperature that does notcause a loss of a component oligonucleotide of a nucleic acidnanostructure containing the functional nucleic acid. In someconfigurations, a melting temperature of a double-stranded nucleic acidcontaining a functional nucleic acid may be designed to be lower than adissociation temperature (e.g., a nucleic acid melting temperature, aligand-receptor dissociation temperature, a covalent bond decompositiontemperature, etc.) for a nucleic acid nanostructure coupled to a solidsupport or a coupling moiety attached to the solid support. For example,a melting temperature of a double-stranded functional nucleic acid of anucleic acid nanostructure may be designed to be at least 5° C., 6° C.,7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16°C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25°C., 26° C., 27° C., 28° C., 29° C., 30° C., 35° C., 40° C., 45° C., 50°C., or more than 50° C. lower than a dissociation temperature of thenucleic acid nanostructure coupled to a solid support or a couplingmoiety of the nucleic acid nanostructure that is attached to the solidsupport.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a capture faceor capture moiety that comprises one or more modifying groups that alteran interaction between the nucleic acid nanostructure and a surface. Analtered interaction between a nucleic acid nanostructure and a surfacemay comprise: 1) increasing the rate or strength of coupling to adesired region of the surface; 2) decreasing the rate or strength ofcoupling to an undesired region of the surface; 3) enhancing thespecificity of coupling to a surface; 4) diminishing non-specificcouplings to a surface; 5) decreasing the strength of interactions(e.g., agglomeration, co-binding) between two or more nucleic acidnanostructures, and 6) combinations thereof. In some configurations, acapture moiety may comprise a modifying moiety, selected from the groupconsisting of an electrically-charged moiety (e.g. a cationic or anionicmoiety), a polar moiety, a non-polar moiety, a ligand moiety that isrecognized by a receptor, a receptor moiety that is recognized by aligand, a magnetic moiety, a steric moiety, an amphipathic moiety, ahydrophobic moiety, and a hydrophilic moiety. In some configurations,the electrically-charged moiety may comprise a single-stranded nucleicacid or a charged polymer (e.g., a cationic or anionic polymer). In someconfigurations, a capture moiety of a nucleic acid nanostructure maycomprise a plurality of single-stranded nucleic acids, where the singlestranded nucleic acids are regions (e.g. tails or loops) of longeroligonucleotides that are hybridized to the nucleic acid nanostructure.In other configurations, a capture moiety of a nucleic acidnanostructure may comprise a plurality of single-stranded nucleic acidsor electrically-charged polymers, where the single stranded nucleicacids are coupled to oligonucleotides that are hybridized to the nucleicacid nanostructure, for example by a covalent linker (e.g., click-typereaction product) or non-covalent linker (e.g., streptavidin-biotincomplex).

Provided herein is a composition comprising: a) a nucleic acidnanostructure (e.g. a structured nucleic acid particle), wherein thenucleic acid nanostructure comprises: i) a display moiety comprising acoupling group that is coupled with, or configured to couple with, ananalyte; and ii) a capture moiety that is coupled with, or configured tocouple with, a surface, wherein the capture moiety comprises a pluralityof first surface-interacting oligonucleotides, and wherein each firstsurface-interacting oligonucleotide of the plurality of firstsurface-interacting oligonucleotides comprises a first nucleic acid thatis coupled with the structured nucleic acid particle and a firstsurface-interacting moiety, wherein the first surface-interacting moietyis coupled with, or configured to form a coupling interaction with, asurface-linked moiety, wherein the capture moiety and the display moietyhave different orientations; and b) an analyte comprising acomplementary coupling group that is coupled with, or configured tocouple with the display moiety of the structured nucleic acid particle.

A nucleic acid nanostructure composition (e.g., a SNAP composition) maycomprise a capture moiety with a plurality of pendant groups thatmediate a coupling interaction with a surface (e.g., a coupling surfaceof a solid support). A pendant group, as set forth herein, may becharacterized by one or more characteristics of: i) comprising anuncoupled terminal moiety or residue, ii) comprising a moiety (e.g., apolymer strand) whose spatial degrees of freedom are not constrained bya coupling interaction with a second moiety of a nucleic acidnanostructure, and iii) comprising a moiety whose average temporalvariations in position relative to a nucleic acid nanostructure exceedan average temporal variation in position of a moiety incorporatedwithin the nucleic acid nanostructure. Without wishing to be bound bytheory, the pendant groups may facilitate multiple properties of anucleic acid nanostructure, including 1) increased specificity ofsurface coupling by the interactions between a capture moiety andsurface-linked moieties on a solid support, 2) increased avidity ofbinding due to a multiplicity of binding interactions between a nucleicacid nanostructure and a coupling surface, 3) tunable binding kineticsbased upon pendant groups added to a nucleic acid nanostructure, 4)tunable binding thermodynamics based upon free energy minimizationbetween a capture moiety and a coupling surface, 5) decreasedinteractions between incidental nucleic acid nanostructure s due tobinding incompatibility of nucleic acid nanostructure capture moieties,and 6) combinations thereof.

FIGS. 40A-40C illustrate SNAP compositions that include pendant groupson the capture moiety of a SNAP. FIG. 40A shows a SNAP 4010 comprisingan upward-oriented display face containing a display moiety 4015 that iscoupled to an analyte 4020 (e.g., a polypeptide). A downward-orientedcapture face of the SNAP 4010 comprises a plurality of pendant groups.Each pendant group comprises an optional linker 4017 and asurface-interacting moiety, such as a surface-interactingoligonucleotide 4018 or a surface-interacting coupling group 4019 (e.g.,a reactive group, a streptavidin, etc.). The SNAP 4010 may be contactedwith a solid support 4000 comprising a coupling surface 4002 and one ormore interstitial regions 4004. The coupling surface 4002 may comprise aplurality of surface-linked groups, in which each surface-linked groupcontains an optional linker 4030 (e.g., a passivating molecule such asPEG) and a surface-linked moiety, such as a complementaryoligonucleotide 4038 or a complementary coupling group 4039 (e.g., acomplementary reactive group, a biotin, etc.). Optionally, a surface maycomprise a mixture of surface-linked groups, in which a first pluralityof surface-linked groups comprises a passivating moiety (e.g., a PEGchain) and no coupling moiety, and a second plurality of surface-linkedgroups comprises a coupling moiety and a passivating moiety (e.g., anoligonucleotide coupled to a PEG chain). FIG. 40B shows a first couplingconfiguration of the SNAP 4010 to the solid support 4000. One or moresurface-interacting oligonucleotides 4018 have hybridized tosurface-linked complementary oligonucleotides 4038, but one or moreother surface-interacting moieties remain unbound. This may suggest thatthe coupled SNAP is not in an energetically favorable binding position.FIG. 40C shows a second coupling configuration of the SNAP 4010 to thesolid support 4000. Each surface-interacting moiety has formed acoupling interaction with a complementary surface-linked moiety. Such aconfiguration may be the most energetically and/or most stable positionfor the SNAP 4010 on the coupling surface 4002.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a capturemoiety that comprises a plurality of oligonucleotides that couple to thenucleic acid nanostructure and provide a plurality of pendant groups, inwhich each pendant group comprises a surface-interacting moiety. Asurface-interacting moiety may form a coupling interaction with asurface-linked moiety on a solid support, thereby coupling a nucleicacid nanostructure comprising the surface-linked moiety to the solidsupport. A nucleic acid nanostructure may comprise a plurality ofoligonucleotides, in which an oligonucleotide of the plurality ofoligonucleotides comprises: a) a first nucleic acid that is configuredto couple to a capture moiety of the nucleic acid nanostructure, and b)a first surface-interacting moiety. In some configurations, the firstsurface-interacting moiety may comprise a second nucleic acid. Forexample, an oligonucleotide of a plurality of oligonucleotides maycomprise a first nucleic acid sequence that is configured to couple to aSNAP and a second nucleic acid sequence that is configured to bind to acomplementary, surface-linked nucleic acid strand of a surface-linkedmoiety by base-pair hybridization. In some cases, the oligonucleotidecontaining the first nucleic acid sequence and the second nucleic acidsequence may further comprise a third nucleic acid sequence that isconfigured to not hybridize to another nucleic acid, for example toprovide flexibility or rigidity to a pendant group as necessary. In someconfigurations, a first surface-interacting moiety may comprise, inaddition to a second nucleic acid or in place of a second nucleic acid,a capture group selected from the group consisting of a reactive group,an electrically-charged group, a magnetic group, and a component of abinding pair. In some configurations, a binding pair may be selectedfrom the group consisting of streptavidin-biotin, SpyCatcher-Spytag,SnoopCatcher-Snooptag, and SdyCatcher-Sdytag. In some configurations, areactive group may be configured to perform a Click-type reaction with asurface-linked moiety. In some configurations, a firstsurface-interacting moiety may comprise a group that is configured toform a non-covalent interaction with a surface-linked moiety, in whichthe interaction is selected from the group consisting of anelectrostatic interaction, a magnetic interaction, a hydrogen bond, anionic bond, a van der Waals bond, a hydrophobic interaction, or ahydrophilic interaction. In particular configurations, a firstsurface-interacting moiety may comprise a nanoparticle selected from thegroup consisting of an inorganic nanoparticle, a carbon nanoparticle, apolymer nanoparticle, and a biopolymer. In some configurations, a firstsurface-interacting moiety may further comprise a linker that couplesthe surface-interacting moiety to a nucleic acid nanostructure. In someconfigurations, the linker may comprise a hydrophobic linker, ahydrophilic linker, or a cleavable linker.

An oligonucleotide comprising a surface-interacting moiety may form aportion of a nucleic acid nanostructure (e.g., a SNAP structure). Anucleic acid nanostructure may comprise a) a scaffold nucleic acidstrand; and b) a plurality of staple nucleic acid strands coupled to thescaffold nucleic acid strand. In some configurations, a plurality ofstaple nucleic acid strands may comprise a first surface-interactingoligonucleotide of a plurality of first surface-interactingoligonucleotides, in which the first surface-interacting oligonucleotidecomprises a surface-interacting moiety. A coupling of a firstsurface-interacting oligonucleotide may form a tertiary structure of anucleic acid nanostructure (e.g., a SNAP). In some configurations, thecapture moiety may comprise a tertiary structure formed by a coupling ofa first surface-interacting oligonucleotide with a nucleic acidnanostructure (e.g., a SNAP). In other configurations, a display moietymay comprise a tertiary structure formed by a coupling of a firstsurface-interacting oligonucleotide with a nucleic acid nanostructure.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a capturemoiety containing a plurality of pendant groups, in which a pendantgroup of the plurality of pendant groups comprises a nucleic acid. Insome configurations, a pendant group may comprise a nucleic acid with anucleotide sequence that comprises no self-complementarity. As such, asurface-interacting oligonucleotide or other nucleic acid can beinhibited from forming a self-hybrid structure under the conditions of acomposition or method set forth herein. For example, a nucleotidesequence of a pendant nucleic acid may comprise a DNA sequence with nomore than 3 deoxyribonucleotide species selected from the groupconsisting of deoxyadenosine, deoxycytosine, deoxyguanosine, anddeoxythymidine (e.g., ACTACCTACAT). In other configurations, a nucleicacid such as a surface-interacting oligonucleotide or pendant group maycomprise a nucleotide sequence that comprises self-complementarity. Forexample, a nucleic acid sequence may form a self-hybrid structure, suchas a double-helix, a stem loop, a pseudoknot, a hairpin or aG-quadruplex under some or all conditions of a composition or method setforth herein. A method set forth herein can be configured such that anucleic acid is in a self-hybrid form in one step but not in anotherstep. For example, in a first step of a method a first nucleic acid canbe in a self-hybrid state to inhibit unwanted hybridization to a secondnucleic acid strand, and in a second step the first nucleic acid can bein a single stranded state or hybridized to a second nucleic acidstrand. In some configurations, a surface-interacting oligonucleotide ofa plurality of surface-interacting oligonucleotides may comprise ahomopolymeric nucleotide sequence selected from the group consisting ofa poly-deoxyadenosine sequence, a poly-deoxycytosine sequence, apoly-deoxyguanosine sequence, or a poly-deoxythymidine sequence. A firstcontiguous sequence of a nucleic acid strand that is configured to formself-complementarity with a second portion of the nucleic acid strandmay comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50contiguous nucleotides. Alternatively or additionally, a firstcontiguous sequence of a nucleic acid strand that is configured to formself-complementarity with a second portion of the nucleic acid strandmay comprise no more than about 50, 45, 40, 35, 30, 25, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less than 3contiguous nucleotides. A first contiguous sequence of a nucleic acidstrand that is configured to form self-complementarity with a secondportion of the nucleic acid strand may be separated from the secondportion of the nucleic acid strand by at least about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 750, 1000,or more than 1000 nucleotides. Alternatively or additionally, a firstcontiguous sequence of a nucleic acid strand that is configured to formself-complementarity with a second portion of the nucleic acid strandmay be separated from the second portion of the nucleic acid strand byno more than about 1000, 750, 500, 400, 300, 200, 100, 95, 90, 85, 80,75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less than 3 contiguousnucleotides.

A pendant nucleic acid portion of a pendant group of asurface-interacting moiety may comprise a particular number of linkednucleotides (e.g., natural nucleotides, modified nucleotides, etc.). Insome cases, a nucleic acid portion of a surface-interacting moiety maycomprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. Alternativelyor additionally, a nucleic acid portion of a surface-interacting moietymay comprise no more than about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 nucleotides.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a capturemoiety with a plurality of pendant groups containing surface-interactingmoieties. A capture moiety may comprise at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100surface-interacting moieties. Alternatively or additionally, a capturemoiety may comprise no more than about 100, 95, 90, 85, 80, 75, 70, 65,60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 surface-interacting moieties.A nucleic acid nanostructure (e.g., a SNAP) may be configured to have anaverage surface density of pendant groups comprising surface-interactingmoieties (e.g., surface-interacting oligonucleotides,surface-interacting reactive groups, etc.). An average surface densityof surface-interacting moieties for a nucleic acid nanostructure may bedetermined by the number of surface-interacting moieties that areconfigured to couple to a coupling surface of a solid support relativeto an effective surface area or footprint of a capture moiety of thenucleic acid nanostructure that couples to the coupling surface. Aneffective surface area of a capture moiety may include a two-dimensionalprojection of the capture moiety onto an effectively planar surface, andmay optionally include additional surface area caused by the maximalextension of one or more pendant groups from the capture moiety of thenucleic acid nanostructure. A footprint of a nucleic acid nanostructuremay comprise a maximum cross-sectional area of a nucleic acidnanostructure or a capture moiety thereof when the nucleic acidnanostructure is coupled to a surface. A capture moiety of a nucleicacid nanostructure (e.g., a SNAP) may have an averagesurface-interacting moiety density of at least 0.0001surface-interacting moieties per square nanometer (nm²), 0.005/nm²,0.001/ nm², 0.05/ nm², 0.01/ nm², 0.05/ nm², 0.1/ nm², 0.5/ nm², 1/ nm²,5/ nm², 10/ nm², or more than 10/nm². Alternatively or additionally, acapture moiety of a nucleic acid nanostructure may have an averagesurface-interacting moiety density of no more than about 10/nm², 5/ nm²,1/ nm², 0.5/ nm², 0.1/ nm², 0.05/ nm², 0.01/ nm², 0.005/ nm², 0.001/nm², 0.0005/ nm², 0.0001/ nm², or less than 0.0001/nm².

A plurality of surface-interacting moieties may be distributed or spacedover a capture moiety of a nucleic acid nanostructure (e.g., a SNAP). Insome configurations, a surface-interacting moiety distribution ordensity is substantially uniform over an effective surface area orfootprint of a capture moiety (e.g., nearly uniform spacing and/ororientation between adjacent surface-interacting moieties). In otherconfigurations, a surface-interacting moiety distribution or density isnot substantially uniform over an effective surface area or footprint ofa capture moiety. For example, a fraction or an entirety of a pluralityof surface-interacting moieties may be located near a central region ofthe capture moiety. In another configuration, a fraction or an entiretyof a plurality of surface-interacting moieties may be located near anouter region of the capture moiety. FIGS. 41A-41B depict SNAPconfigurations with differing SNAP distributions. FIG. 41A depicts aSNAP 4110 that is coupled to an analyte 4120 and contains a plurality ofsurface-interacting moieties 4118 on a capture moiety, in which theplurality of surface-interacting moieties is distributed toward theouter edges of the capture moiety face. FIG. 41B depicts a SNAP 4110that is coupled to an analyte 4120 and contains a plurality ofsurface-interacting moieties 4118 on a capture moiety, in which theplurality of surface-interacting moieties is distributed toward thecentral portion of the capture moiety face.

In some configurations, a nucleic acid nanostructure (e.g., a SNAP) maycomprise a capture moiety comprising more than one type ofsurface-interacting moiety. A capture moiety may comprise more than onetype of surface-interacting moiety to increase the specificity ofbinding location for a nucleic acid nanostructure. For example, a SNAPmay comprise a plurality of surface-interacting oligonucleotides and oneor more surface-interacting reactive groups. In a particular example,such a SNAP may be contacted with a coupling surface comprising a highsurface density of complementary oligonucleotides and a low surfacedensity of complementary reactive groups, in which binding interactionsbetween surface-interacting oligonucleotides and complementaryoligonucleotides keep the SNAP coupled near the coupling surface until acovalent binding interaction can form between the surface-interactingreactive group and the relatively rare, surface-linked complementaryreactive group. A nucleic acid nanostructure may interact with a surfacethrough a combination of types of interactions, such as through twodiffering non-covalent interactions (e.g., nucleic acid hybridizationand an electrostatic interaction, etc.), two differing covalentinteractions (e.g., two bioorthogonal Click-type reactions), or acombination of a covalent interaction and a non-covalent interaction(e.g., a covalent interaction and nucleic acid hybridization, a covalentinteraction and an electrostatic interaction, a covalent interactionwith nucleic acid hybridization and electrostatic interactions, etc.).

In another aspect, provided herein is a composition comprising: a)nucleic acid nanostructure (e.g., a SNAP), wherein the nucleic acidnanostructure comprises: i) a display moiety that is coupled with, orconfigured to couple with, an analyte; and ii) a capture moiety that iscoupled with, or configured to couple with a coupling surface, whereinthe capture moiety comprises a plurality of oligonucleotides, andwherein each oligonucleotide of the plurality of oligonucleotidescomprises a surface-interacting moiety; b) an analyte coupled with thedisplay moiety; and c) a solid support comprising the coupling surface,wherein the surface comprises one or more surface-linked moieties, andwherein a surface-interacting moiety of the plurality ofsurface-interacting moieties is coupled with a surface-linked moiety ofthe one or more surface-linked moieties.

A nucleic acid nanostructure composition (e.g., a SNAP composition), asset forth herein, may further comprise a separating group. A separatinggroup may comprise a molecule, linker, or nucleic acid nanostructure(e.g., a display SNAP or a structural SNAP) that is configured to createa separation or gap between an analyte and a surface or a portion of anucleic acid nanostructure (e.g., a display face or moiety, a captureface or moiety). FIG. 29 illustrates a profile view of a SNAP complexcomprising an analyte with various possible separation gaps labeled. TheSNAP complex may comprise capture utility SNAPs 2910, 2911 and 2912 thatcouple the complex to a solid support 2900. A display SNAP 2930 iscoupled to a structural utility SNAP 2920 that is coupled to the captureutility SNAP 2911. An analyte 2940 is coupled to the display SNAP 2930.A separation gap may be measured from the analyte to a surface or SNAP.Some possible separation gaps may include the gap from the center ofanalyte 2940 to the solid support 2900 (g₁), to the top face of thecapture utility SNAPs 2910 (g₂) or the top face of the display SNAP 2930(g₃); the gap between the external surface of analyte 2940 and thesurface of solid support 2900 (g₄); the gap between the external surfaceof analyte 2940 and the face of capture utility SNAP 2910 (g₅); or thegap between the external surface of analyte 2940 and the face of thedisplay SNAP 2930 (g₆). FIGS. 3A-3D illustrate a SNAP 300 comprising apolyvalent linker 320 that creates an average separation gap between ananalyte 310 and the upper face of the SNAP 300. If the SNAP 300 iscoupled to a solid support 330, the analyte 310 will also have anaverage separation gap with the solid support 330. In someconfigurations, a separating group may comprise a rigid separating groupselected from the group comprising a polymer linker, a nucleic acidlinker, and a nanoparticle linker. In some specific configurations, thenucleic acid linker comprises a tertiary structure (e.g., a DNA doublehelix). In other configurations, the separating group comprises aflexible linker. A separation gap may have a characteristic average,maximum of minimum dimension. The average, maximum or minimum dimensionof a separation gap can be at least about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm,6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm,17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60nm, 70 nm, 80 nm, 90 nm, 100 nm, or more than 100 nm. Alternatively oradditionally, the average, maximum or minimum dimension of a separationgap can be no more than about 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm,45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15nm, 14 nm, 13 nm, 12 nm, 11 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4nm, 3 nm, 2 nm, 1 nm, or less than 1 nm.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a plurality ofnucleic acids (e.g., scaffold strands, a plurality of oligonucleotides)that form stable hybridized structures through complementary base pairbinding. The stability of specific hybridized structures may becharacterized through routine methods, such as by degree ofcomplementarity or estimated or measured secondary structure meltingtemperature. A stability (e.g., a melting temperature) may be predictedby a software package, such as CADNANO, ATHENA, or DAEDALUS. Ahybridized nucleic acid structure may have a characterized meltingtemperature of at least about 50° C., 51° C., 52° C., 53° C., 54° C.,55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C.,64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C.,73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C.,82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C.,or more than 90° C. Alternatively or additionally, a hybridized nucleicacid structure may have a characterized melting temperature of no morethan about 90° C., 89° C., 88° C., 87° C., 86° C., 85° C., 84° C., 83°C., 82° C., 81° C., 80° C., 79° C., 78° C., 77° C., 76° C., 75° C., 74°C., 73° C., 72° C., 71° C., 70° C., 69° C., 68° C., 67° C., 66° C., 65°C., 64° C., 63° C., 62° C., 61° C., 60° C., 59° C., 58° C., 57° C., 56°C., 55° C., 54° C., 53° C., 52° C., 51° C., 50° C., or less than 50° C.

A nucleic acid nanostructure (e.g., a SNAP) or a face of a nucleic acidnanostructure (e.g., a display face, a capture face) may have acharacteristic dimension (e.g., length, width, radius). A characteristicdimension may include any characterizing measure pertaining to the groupor probe size, such as length, width, height, radius, circumference,etc. A nucleic acid nanostructure or a face of a nucleic acidnanostructure may have a characteristic dimension of at least about 5nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 120nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or more than 1000nm. Alternatively or additionally, a nucleic acid nanostructure or aface of a nucleic acid nanostructure may have a characteristic dimensionof no more than about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180 nm, 160 nm, 140 nm,120 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm,55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5nm, or less than 5 nm.

A nucleic acid nanostructure (e.g., a SNAP) may be coupled to, orconfigured to couple to, one or more analytes. A nucleic acidnanostructure may comprise one or more display faces or display moietiesthat are coupled to, or configured to couple to, one or more analytes. Anucleic acid nanostructure may be coupled to one or more analytes. Anucleic acid nanostructure may comprise one or more display faces ordisplay moieties that are coupled to one or more analytes. A nucleicacid nanostructure display face or display moiety may comprise one ormore functional groups or moieties that are configured to couple to ananalyte. When multiple functional groups are present, the functionalgroups can be the same type as each other, or alternatively, differentfunctional groups can be present. A nucleic acid nanostructure maycomprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, or more than 100 functional groups or moieties that areconfigured to couple to an analyte. Alternatively or additionally, anucleic acid nanostructure may comprise no more than about 100, 95, 90,85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than about 2functional groups or moieties that are configured to couple to ananalyte.

A plurality of nucleic acid nanostructures (e.g., SNAPs) and a pluralityof analytes may be coupled in a fixed molecular ratio. The ratio ofanalyte to nucleic acid nanostructures may be calculated as an averageratio. The analyte:nanostructure ratio may follow some quantifiabledistribution, such as a Poisson distribution, binomial distribution,beta-binomial distribution, hypergeometric distribution, or bimodaldistribution. In some configurations, there may be, on average, morethan one analyte coupled to a nucleic acid nanostructure. In someconfigurations, there may be, on average, more than one nucleic acidnanostructure coupled to an analyte. A plurality of analyte-couplednucleic acid nanostructures may have an average analyte:nanostructureratio of no more than about 100:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1,4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20,1:25, 1:50, 1:100, or less than 1:100. Alternatively or additionally, aplurality of analyte-coupled nucleic acid nanostructures may have anaverage analyte:nanostructure ratio of at least about 1:100, 1:50, 1:25,1:20, 1:15, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 3:1, 4:1,5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 100:1, or more than 100:1.

A plurality of nucleic acid nanostructures (e.g., SNAPs) may becharacterized by an occupancy ratio. An occupancy ratio may be definedas the fraction of nucleic acid nanostructures with at least one coupledanalyte. The nucleic acid nanostructure occupancy ratio may becontrolled to provide a desired occupancy (such as a maximum occupancy)by increasing the relative ratio of analytes to nucleic acidnanostructures during analyte coupling. The nucleic acid nanostructureoccupancy ratio may be controlled to minimize the number of nucleic acidnanostructures with more than one analyte by, for example, reducing theconcentration of analyte relative to nucleic acid nanostructures duringanalyte coupling. For example, a composition of SNAPs with 70% of theSNAPs being coupled to one or more analytes would have an occupancyratio of 0.7. Occupancy ratio may be determined by an appropriateanalytical technique, such as fluorescent microscopy or spectroscopicanalysis. A plurality of nucleic acid nanostructures may have anoccupancy ratio of at least about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4. 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95,0.96, 0.97, 0.98, 0.99, or more than 0.99. Alternatively oradditionally, a plurality of nucleic acid nanostructures may have anoccupancy ratio of no more than about 0.99, 0.98, 0.97, 0.96, 0.95, 0.9,0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25,0.2, 0.15, 0.1, 0.05, 0.01, or less than about 0.01.

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, mayfurther comprise a capture face. The capture face may be configured tofacilitate an interaction between a surface or an interface, such as abinding interaction or a phase separation interaction. A surface may beany solid and/or rigid boundary where the nucleic acid nanostructure issubstantially inhibited from, or cannot, transfer orthogonally throughthe solid and/or rigid boundary. An interface may refer to a non-solidor deformable boundary where the nucleic acid nanostructure can transferorthogonally through the non-solid or deformable boundary. A surface maycomprise a surface of a solid material such as a metal, metal oxide,ceramic, glass, polymer, or semiconductor. An interface may comprise anair/liquid or liquid/liquid phase boundary. Exemplary interfaces mayinclude an air/water interface, or a water/oil interface such as anoil-in-water or water-in-oil emulsion. A capture face or capture moietymay be configured to form a reversible or irreversible interaction witha surface. For example, a capture face of a SNAP may comprise one ormore single-stranded nucleic acid strands that are configured tohybridize to complementary single-stranded nucleic acids that aredisplayed on a surface, thereby reversibly coupling the SNAP to thesurface. In another example, a capture face of a SNAP may comprise oneor more click-type reaction groups that are configured to covalentlybond to complementary click-type reaction groups that are displayed on asurface, thereby irreversibly coupling the SNAP to the surface. In someconfigurations, a nucleic acid nanostructure (e.g., a SNAP) may comprisea capture face comprising a first moiety and a second moiety, where thefirst moiety is configured to reversibly couple to a surface and secondmoiety is configured to irreversibly couple to a surface. In some cases,a nucleic acid nanostructure may be configured to provide a temporaryassociation with a solid support. For example, a SNAP may be configuredto reversibly couple an analyte (e.g., by an oligonucleotide hybridizedto the SNAP structure), then bind to a surface of the solid supporttemporarily, thereby permitting the analyte to be transferred to ananalyte-coupling moiety on the surface (e.g., a complementaryoligonucleotide, a Click-type reactive group, etc.). After the analytehas been transferred to the surface, the SNAP may be dissociated andoptionally reused to transfer a second analyte to the solid support.

A nucleic acid nanostructure (e.g., a SNAP) may interact with a surfaceor interface by an interaction that associates the nucleic acidnanostructure with the surface or interface. A nucleic acidnanostructure may associate with a surface or an interface by a bindinginteraction such as an electrostatic interaction, magnetic interaction,covalent bond, or non-covalent bond (e.g., hydrogen bonding, nucleicacid base pair binding). A nucleic acid nanostructure may comprise oneor more faces that are configured to effect a phase separation at aphase boundary. For example, a SNAP may comprise a first face comprisinga plurality of hydrophobic moieties and a second face comprising aplurality of hydrophilic moieties, where the SNAP is configured tobecome associated to a phase boundary by segregation of the first faceinto a more hydrophobic phase.

FIGS. 4A-4G show various configurations of a SNAP interacting with asurface or interface. FIG. 4A illustrates a SNAP 410 coupled to ananalyte 420 interacting with a surface 430 via an electrostaticinteraction. A SNAP may comprise a negatively charged capture face 412that may be attracted to a positively-charged surface 430, for example asurface 430 functionalized with positively-charged functional groups432. The negative charge of the SNAP may be due to one or both of thenegative charges present in phosphodiester backbone of nucleic acid ornegatively charged moieties conjugated to the SNAP. FIG. 4B illustratesa SNAP 410 coupled to an analyte 420 (e.g., a polypeptide) interactingwith a surface 430 via a magnetic interaction. The SNAP may comprise acapture face 412 comprising a plurality of magnetic groups (e.g.,paramagnetic particles conjugated to the SNAP) that may be attracted toa surface 430, for example a surface 430 comprising a plurality ofoppositely-polarized magnetic groups 438. FIG. 4C illustrates a SNAP 410coupled to an analyte 420 (e.g., a polypeptide) interacting with asurface 430 by a non-covalent binding interaction between complementaryoligonucleotides. The SNAP 410 comprises a capture face 412 comprising aplurality of oligonucleotides 414 that hybridize with a plurality ofcomplementary oligonucleotides 434 that are coupled to the surface 430.FIG. 4D illustrates a SNAP 410 coupled to an analyte 420 (e.g., apolypeptide) that is covalently conjugated to a surface 430. A covalentlinkage 435 may form between complementary reactive groups on thesurface 430 and the capture face 412 of the SNAP 410, such as clickreaction groups (e.g., methyltetrazine-transcyclooctylene,azide-dibenzocylooctyne, etc.). In some configurations, the SNAP 410 maycomprise a plurality of reactive groups on the capture face 412 that areconfigured to form covalent linkages 430.

FIGS. 4E-4F depict configurations of SNAPs interacting with an interface(e.g., water/air or water/oil). A SNAP may associate with an interfaceby a phase separation interaction. FIG. 4E depicts a SNAP 410 coupled toan analyte 420 comprising a capture face 412 containing a plurality ofhydrophobic groups 417 (e.g., lipids). The presence of the hydrophobicgroups 417 associates the SNAP 410 with an interface 440 that formsbetween a non-aqueous phase 444 and an aqueous phase 448. Thehydrophobic groups 417 may preferentially migrate into the non-aqueousphase 444 while the more hydrophilic SNAP 410 and analyte 420 may remainin the aqueous phase 448. FIG. 4F depicts an alternative configurationof an interface-associating SNAP 410. FIG. 4F depicts a SNAP 410 coupledto an analyte 420 comprising a capture face 412 containing a pluralityof hydrophobic groups 417. The SNAP is further configured such that thecapture face 412 is also the display face of the SNAP. The presence ofthe hydrophobic groups 417 associates the SNAP 410 with an interface 440that forms between a non-aqueous phase 444 and an aqueous phase 448. Thehydrophobic groups 417 and the analyte 420 may preferentially migrateinto the non-aqueous phase 444 while the more hydrophilic SNAP 410 mayremain in the aqueous phase 448. The configuration of FIG. 4F may beadvantageous for the display of hydrophobic analytes (e.g., membraneproteins, inorganic nanoparticles).

FIG. 4G depicts a configuration of a SNAP 410 coupled to an analyte 420interacting with a surface 430 by an ion-mediated coupling interaction.A SNAP may comprise a negatively charged capture face 412 that may beattracted to a surface 430, for example a surface 430 functionalizedwith negatively-charged functional groups 433. In other configurations,the surface material may possess an inherent negative charge. Thenegative charge of the SNAP 410 may be due to the negative chargespresent in phosphodiester backbone of nucleic acid or due to negativelycharged moieties conjugated to the SNAP. The inherent repulsion betweenthe capture face 412 of the SNAP 410 and the negatively-chargedfunctional groups 433 may be overcome by the complexing or layering ofpositively-charged ions 450 to for an ion-mediating layer between theSNAP 410 and the surface 430. The skilled person will readily recognizethat ion-mediated interactions may be modified for other situations,such as mediating positive-positive charge interactions, or varying thestrength of positive-negative charge interactions. Deposition of SNAPsat a surface by an ion-mediated charge interaction may occur in thepresence of a particular monatomic ion, polyatomic ion, monovalent ion,polyvalent ion, metal ion, or non-metal ion, such as H⁺, Li⁺, Na⁺, K⁺,Rb⁺, Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Al³⁺, Ag⁺, Zn²⁺, Fe²⁺, Fe³⁺, Cu⁺,Cu²⁺, H⁻, F⁻, Cl⁻, Br⁻, I⁻, O²⁻, S²⁻, N³⁻, P³⁻, B(OH)₄ ⁻, C₂H₅O⁻,CH₃COO⁻, C₆H₅COO⁻, C₆H₅O₇ ³⁻, CO₃ ²⁻, C₂O₄ ²⁻, CN⁻, CrO₄ ²⁻, Cr₂O₇ ²⁻,HCO₃ ⁻, HPO₄ ²⁻, H₂PO₄ ⁻, HSO₄ ⁻, MnO₄ ²⁻, MnO₄ ⁻, NH₂ ⁻, O₂ ²⁻, OH⁻,SH⁻, SCN⁻, SiO₄ ²⁻, S₂O₃ ²⁻, C(NH₂)³⁺, NH₄ ⁺, PH₄ ⁺, H₃O⁺, H₂F⁺, C₅H₅O⁺,Hg₂ ²⁺, and combinations thereof. FIG. 4H depicts a configuration of aSNAP 410 coupled to an analyte 420 interacting with a surface 430 by aparticle-mediated coupling interaction. A SNAP may comprise apositively-charged capture face 412 (e.g., comprising one or moreaminated capture moieties) that may be inherently repulsed by a surface430, for example a surface 430 functionalized with positively-chargedfunctional groups 433 (e.g., aminated silanes). An intermediatenegatively-charged particle 460 may facilitate an interaction betweenthe SNAP 410 and the surface 430 by passivating the surface positivecharge and providing a negative charge that electrostatically couplesthe positively-charged capture face 412 of the SNAP 410.Negatively-charged particles 460 may include carboxylated inorganicnanoparticles (e.g., carboxylated gold nanoparticles, carboxylatedsilver nanoparticles, etc.) or carboxylated organic nanoparticles (e.g.,carboxylated dextran nanoparticles, carboxylated polystyrene particles,etc.).

In some configurations, a nucleic acid nanostructure (e.g., a SNAP) maybe structured to inhibit or avoid forming a charge-mediated interaction.Nucleic acid nanostructures may be non-specifically attracted to areasof a surface where deposition is not supposed to occur due tocharge-mediated interactions, for example, by ionic components of adeposition buffer. A nucleic acid nanostructure may be configured todisplay ligands or other groups on a capture face or capture moiety thatdisrupt unwanted interactions. For example, a SNAP may comprise one ormore single-stranded nucleic acids (e.g., pendant tails ofoligonucleotides that partially hybridize to the SNAP structure) thatdisrupt the formation of charge-mediated interactions. In anotherexample, a SNAP may comprise a capture moiety containing one or moreoligonucleotides, where each oligonucleotide comprises a modifiednucleotide that is configured to disrupt the formation of acharge-mediated interaction. The modified nucleotides may be chemicallyhomogeneous (e.g., same charge, same structure, same polarity, etc.) ormay be chemically heterogeneous.

A capture face of a nucleic acid nanostructure (e.g., a SNAP) may beconfigured to mediate the association between the nucleic acidnanostructure and a surface or interface. The configuration of a nucleicacid nanostructure may determine the strength of an association betweenthe nucleic acid nanostructure and a surface or interface. A nucleicacid nanostructure may have a reversible or irreversible associationwith a surface or interface. An irreversible association between anucleic acid nanostructure and a surface or interface may be formed bycovalent bonding or very strong non-covalent interaction(s) (e.g.,streptavidin-biotin). A reversible association between a nucleic acidnanostructure and a surface or interface may be formed by a weakerinteraction such as an electrostatic interaction, magnetic interaction,or hydrogen bonding. A reversible association may be stable until it isdisrupted, for example by the introduction of a denaturant or salt, orthe cleavage of a photolinker.

The size and or conformation of a nucleic acid nanostructure captureface may affect the strength of an association between a nucleic acidnanostructure and a surface or interface. A smaller interaction regionbetween a capture face and a surface or interface may facilitate aweaker interaction between a nucleic acid nanostructure and the surfaceor interface. A capture face or capture moiety may comprise one or moretertiary nucleic acid structures that form interactions with a surface,such as an electrostatic interaction. Increased size or number oftertiary structures in a capture face or capture moiety may increase thestrength of an interaction with a surface. For example, increased size,increased quantity, or increased local density of nucleic acid tertiarystructures in a capture moiety may increase the strength of anelectrostatic interaction between the capture moiety and a surface dueto an increased number of negatively-charged phosphodiester groups inthe nucleic acid backbones of each tertiary structure. FIGS. 5A-5Ddepict various configurations of SNAPs with differing capture face sizesand/or conformations. FIGS. 5A and 5B depict tapered SNAP structureswith differing two-dimensional projections between the display face andthe capture face. FIG. 5A depicts a SNAP 510 that is bound to a surface530. The SNAP comprises a larger display face 520 comprising a displaymoiety 522. The SNAP also comprises a capture face 540 whose area issmaller than the area of the display face 520. The capture face 540forms a small interaction region 545 with the surface 530, possiblyleading to a weaker association between the SNAP 510 and the surface530. FIG. 5B depicts a SNAP 510 that is bound to a surface 530. The SNAPcomprises a smaller display face 520 comprising an analyte conjugationsite 522. The SNAP also comprises a capture face 540 whose area islarger than the area of the display face 520. The capture face 540 formsa large interaction region 545 with the surface 530, optionally leadingto a stronger association between the SNAP 510 and the surface 530. FIG.5C depicts a SNAP 510 comprising a non-planar capture face 540 thatassociates the SNAP 510 with a surface 530. The SNAP comprises a largerdisplay face 520 containing a display moiety 522. Due to the non-planarcapture face, the SNAP forms a smaller interaction region 545 with thesurface 530, optionally leading to a weaker association between the SNAP510 and the surface 530. FIG. 5D depicts a SNAP 510 comprising anon-planar capture face 540 that associates the SNAP 510 with anon-planar surface 535. The SNAP comprises a display face 520 containinga capture moiety 522. Due to the shape complementarity between thecapture face 540 and the non-planar surface 535, the SNAP forms a largerinteraction region 545 with the surface 535, possibly leading to astronger association between the SNAP 510 and the surface 535.Accordingly, the size and/or shape of a nucleic acid nanostructure(e.g., a SNAP) capture face can be useful for orienting the nucleic acidnanostructure on a surface. The surface can be patterned withinteraction regions to provide further control over location and/ororientation of nucleic acid nanostructures on the surface. For example,a hexagonal array of nucleic acid nanostructures can be formed byattachment of the nanostructures to a surface having a hexagonal patternof interaction regions, wherein the interaction regions are separated byinterstitial regions that are inert to binding the nanostructures.Moreover, engineering the size and/or shape for one or both of a surfaceand a plurality of nucleic acid nanostructures can provide for controlover the arrangement of the nucleic acid nanostructures into an array.Accordingly, a user can achieve a desired density of nucleic acidnanostructures in the array, average spacing of nucleic acidnanostructures in the array, minimal separation between adjacent nucleicacid nanostructures in the array or maximum separation between adjacentnucleic acid nanostructures in the array. As such, analytes that areconjugated to nucleic acid nanostructures will also be arrangedaccordingly.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a capture facethat forms a smaller interaction region than its two-dimensionalprojection. FIG. 6 depicts views of the bottom surface and top surfaceof a rectangular-shaped SNAP 600. The correspondence of edges betweenthe top view and bottom view are indicated by the dashed lines. The SNAP600 comprises a capture face 610 that is configured to only contact asurface or interface (not shown) around the perimeter of the SNAP 600.The SNAP further comprises a display face 620 comprising a displaymoiety 622. The display face 620 occupies the full area of the top faceof the SNAP 600. The configuration depicted in FIG. 6 would limit thesize and/or strength of an association between the SNAP 600 and asurface or interface while maximizing the available area for analytedisplay. The skilled person will readily recognize that theconfiguration depicted in FIG. 6 could be reconfigured to increase ordecrease the sizes of the capture faces 610 and displaying surface 620by altering the structured nucleic acid components that constitute theSNAP 600.

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, maycomprise a utility face or utility moiety comprising one or moremodifying moieties. In some configurations, a utility face may compriseall or portions of another face, such as a display face or a captureface. Modifying moieties may be added to a capture face or capturemoiety to alter the characteristics of the surface while mediating anassociation between a nucleic acid nanostructure and a surface, anucleic acid nanostructure and an interface, a first nucleic acidnanostructure and a second nucleic acid nanostructure, or a nucleic acidnanostructure and a coincident molecule (e.g., an affinity reagent, afluorophore, etc.). Modifying moieties may be attached covalently ornon-covalently. Modifying moieties may be coupled to a nucleic acidnanostructure before, during, or after assembly of the nanostructure.Utility face modification groups may include electrically-chargedmoieties, magnetic moieties, steric moieties, amphipathic moieties,optical moieties (e.g., reflective materials, absorptive materials),hydrophobic moieties, and hydrophilic moieties. Electrically-chargedmoieties may include functional groups that may carry an intrinsicpositive or negative charge, or may carry a charge under dissociatingconditions (e.g., carboxylic acids, nitrates, sulfones, phosphates,phosphonates, etc.). Magnetic moieties may include paramagnetic,diamagnetic, and ferromagnetic particles such as nanoparticles (e.g.,gadolinium, manganese, iron oxide, bismuth, gold, silver, cobaltnanoparticles, etc.). Steric moieties may include polymers andbiopolymers (e.g., PEG, PEO, dextran, sheared nucleic acids).Amphipathic moieties may include phospholipids (e.g., phosphatidic acid,phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylinositol phosphate,phosphatidylinositol biphosphate, phosphatidylinositol triphosphate,ceramide phosphorylcholine, ceramide phophorylethanolamine, ceramidephosphoryllipid), glycolipids (e.g., glyceroglycolipids,sphingoglycolipids, rhamnolipids, etc.), and sterols (e.g., cholesterol,campesterol, sitosterol, stigmasterol, ergosterol, etc.). Hydrophobicmoieties may include steroids (e.g., cholesterol), saturated fatty acids(e.g., caprylic acid, capric acid, lauric acid, myristic acid, palmiticacid, stearic acid, arachidic acid, behenic acid, lignoceric acid,cerotic acid, etc.), and unsaturated fatty acids (e.g., myristoleicacid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid,vaccenic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid,erucic acid, docosahexanenoic acid, etc.). Hydrophilic compounds mayinclude charged molecules and polar molecules (e.g., glycols,cyclodextrins, cellulose, polyacrylamides, etc.).

In some configurations, a nucleic acid nanostructure (e.g., a SNAP) maycomprise a utility face or utility moiety comprising one or moreextendable nucleic acid (e.g., a nucleic acid primer) or extendednucleic acids (e.g. an extended nucleic acid primer). A primer or otherextendable nucleic acid terminus can be hybridized to a template strandto direct polymerase-based extension. However, extension need notinvolve addition of nucleotides by a template-directed polymerase, forexample, instead involving nucleotide addition by a terminaldeoxynucleotidyl transferase or oligonucleotide addition by a ligase.Optionally, some or all nucleic acid termini in the nucleic acidnanostructure, other than a given primer that is to be extended, can benon-extendable, for example, due to the presence of a 5′ or 3′ extensionblocking moiety. Accordingly, extension can selectively occur at thegiven primer instead of at the other termini. Exemplary extensionblocking moieties include, but are not limited to, those used in nucleicacid sequencing-by-synthesis reactions such as reversible terminators.Reversible terminator moieties can be particularly useful since they canbe present at a first nucleic acid to prevent its extension duringextension of a second nucleic acid terminus, and then removed from thefirst terminus to render it extendable.

An extended nucleic acid may be configured to occupy a volumesurrounding a nucleic acid nanostructure and/or exclude other molecules(e.g., other SNAPs, analytes, etc.) from approaching or contacting thenucleic acid nanostructure. An extended nucleic acid may comprise asingle-stranded nucleic acid strand, a double-stranded nucleic acidstrand, or a combination thereof. An extended nucleic acid may comprisea secondary structure (e.g., a helical structure). An extended nucleicacid may comprise a region of random or disordered structure. Anextended nucleic acid strand may incorporate modified or non-naturalnucleotides, or other linking moieties. An extended nucleic acid may beformed by a method such as terminal deoxynucleotidyl transferase (TdT)polymerization. Methods of forming extended nucleic acids are describedin Yang, et al. Angewandte Chemie Int. Ed., 10.1002/anie.202107829,(2021), which is herein incorporated by reference in its entirety. Anextended nucleic acid may have a sequence comprising at least about 100,200, 300, 400, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000,10000, 15000, 20000, or more than 20000 nucleotides. Alternatively oradditionally, an extended nucleic acid may have a sequence comprising nomore than about 20000, 15000, 10000, 5000, 4000, 3000, 2500, 2000, 1500,1000, 750, 500, 400, 300, 200, 100, or less than 100 nucleotides. Anextended nucleic acid may have a length, in an extended or condensedstate (e.g., coiled, self-hybridized, etc.), of at least about 10nanometers (nm), 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm,100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm,400 nm, 450 nm, 500 nm, or more than 500 nm. Alternatively oradditionally, an extended nucleic acid may have a length, in an extendedor condensed state (e.g., coiled, self-hybridized, etc.), of no morethan about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180nm, 160 nm, 140 nm, 120 nm, 100 nm, 90 nm, 80. nm, 70 nm, 60 nm, 50 nm,40 nm, 30 nm, 20 nm, 10 nm, or less than 10 nm.

A utility face or utility moiety of a nucleic acid nanostructure (e.g.,a SNAP) may comprise one or more modifying moieties. A utility face of anucleic acid nanostructure may comprise at least about 10, 50, 100, 500,1000, 5000, 10000, 50000, 100000, 50000, 1000000, or more than 1000000modifying groups. Alternatively or additionally, a utility face of anucleic acid nanostructure may comprise no more than about 1000000,500000, 100000, 50000, 10000, 5000, 1000, 500, 100, 50, 10, or less than10 modifying groups.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a utility facewith a characteristic density of modifying moieties. The modifyingmoiety density may refer to an average or localized area density ofmodifying moieties on a nucleic acid nanostructure utility face. Autility face of a nucleic acid nanostructure may have a modifying moietydensity of no more than about 1 group/nm², 1 group/10 nm², 1 group/100nm², 1 group/1000 nm², 1 group/10000 nm², 1 group/100000 nm², 1group/1000000 nm², or less than 1 group/1000000 nm². Alternatively oradditionally, a utility face of a nucleic acid nanostructure may have amodifying moiety density of at least about 1 group/1000000 nm², 1group/100000 nm², 1 group/10000 nm², 1 group/1000 nm², 1 group/100 nm²,1 group/10 nm², 1 group/nm², or more than 1 group/nm².

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, maycomprise one or more detectable labels, for example, at a utility faceof the nanostructure. A detectable label may comprise a group that isconfigured to provide or transmit a signal. A detectable label mayprovide or transmit a signal in real time (e.g., a fluorophore, aradiolabel) or at a later time (e.g., a barcode). A detectable label maycomprise a detectable label selected from the group consisting of afluorescent group, a luminescent group, a radiolabel, an isotope, and abarcode. Any of a wide variety of fluorescent labels known in the artmay be used to label the probes. In some cases, the fluorescent labelmay be a small molecule. In some cases, the fluorescent label may be aprotein. In some cases, the fluorescent label may be a nanoparticle(e.g., a quantum dot, a fluorescently-labeled polymer nanoparticle,etc.). Fluorescent labels may include labels that emit in theultraviolet spectrum, visible spectrum, or infrared spectrum. In somecases, the fluorescent molecule may be selected from the groupconsisting of FITC, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor®488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor®568, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor®750, Pacific Blue, Coumarin, BODIPY FL, Pacific Green, Oregon Green,Cy3, Cy5, Pacific Orange, TRITC, Texas Red, R-Phycoerythrin, andAllophycocyanin (APC). In some cases, the label may be an Atto dye, forexample Atto 390, Atto 425, Atto 430, Atto 465, Atto 488, Atto 490, Atto495, Atto 514, Atto 520, Atto 532, Atto 540, Atto 550, Atto 565, Atto580, Atto 590, Atto 594, Atto 610, Atto 611, Atto 612, Atto 620, Atto633, Atto 635, Atto 647, Atto 655, Atto 680, Atto 700, Atto 725, Atto740, Atto MB2, Atto Oxa12, Atto Rho101, Atto Rho12, Atto Rho13, AttoRho14, Atto Rho3B, Atto Rho6G, or Atto Thio12. A wide range of effectivefluorescent labeling groups may be commercially available from theMolecular Probes division of ThermoFisher Scientific and are generallydescribed in the Molecular Probes Handbook (11th Edition) which ishereby incorporated by reference. Detectable labels may also includeintercalation dyes, such as ethidium bromide, propidium bromide, crystalviolet, 4′,6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin D(7-AAD), Hoescht 33258, Hoescht 33342, Hoescht 34580, YOYO-1, DiYO-1,TOTO-1, DiTO-1, or combinations thereof.

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, maycomprise a three-dimensional structure. A nucleic acid nanostructure maycomprise a plurality of faces, including a display face, a binding face,and additional utility faces. In some configurations, utility faces maybe located on the regions of a nucleic acid nanostructure thatconstitute a height or depth of the nucleic acid nanostructure. Autility face may be utilized for any of a variety of purposes, includingcoupling a nucleic acid nanostructure to other structures, or providingspacing between a nucleic acid nanostructure and other structures ormolecules. A utility face may comprise one or more modifying groups.Utility face modifying groups may be attached covalently ornon-covalently. Utility face modifying groups may be coupled to anucleic acid nanostructure before, during, or after assembly of thenanostructure. Utility face modifying groups may includeelectrically-charged moieties, magnetic moieties, steric moieties,hydrophobic moieties, hydrophilic moieties, and coupling groups.Coupling groups may comprise any groups that are configured to couple anucleic acid nanostructure to a solid support or to another molecule,such as another nucleic acid nanostructure. Coupling groups may includecovalent coupling groups and non-covalent coupling groups. Covalentcoupling groups may include chemically reactive species such as clickreaction groups and cross-linking molecules. Cross-linking molecules mayinclude chemical cross-linking molecules and photo-initiatedcross-linking molecules. Non-covalent coupling groups may includebinding pairs (e.g., streptavidin-biotin) and nucleic acids configuredto base-pair with complementary nucleic acids on other molecules. Anucleic acid nanostructure (e.g., a SNAP), molecule that is to beconjugated to a nucleic acid nanostructure, or solid support that is tobe conjugated to a nucleic acid nanostructure can include any of avariety of coupling groups such as those set forth in U.S. patentapplication Ser. No. 17/062,405 or WO 2019/195633 A1, each of which isincorporated herein by reference. A utility face of a nucleic acidnanostructure may comprise one or more steric groups that hinder othermolecules from approaching within a proximity of the nucleic acidnanostructure, as determined by the size of the one or more stericgroups.

A nucleic acid nanostructure (e.g., a SNAP) may comprise one or morecoupling faces or coupling moieties. A utility face or a utility moietymay comprise one or more functional groups or moieties that areconfigured to couple a first nucleic acid nanostructure to a secondnucleic acid nanostructure. Coupling moieties may include those setforth herein, for example in the context of utility faces. Couplingsbetween nucleic acid nanostructures (e.g., a display SNAP and a spacerSNAP) or between nucleic acid nanostructure complexes may be formed bythe reversible or irreversible binding of complementary sets of couplingmoieties on each pair-forming nucleic acid nanostructure. Reversiblebinding of complementary nucleic acid nanostructures may occur via anon-covalent bond (e.g., nucleic acid hybridization, hydrogen bonding)or a thermodynamically-reversible covalent bond (e.g., a peroxide bond,a disulfide bond). A nucleic acid nanostructure or complex thereof maycomprise one or more coupling groups that are configured to couple withone or more complementary coupling moieties on a second nucleic acidnanostructure or complex thereof. A nucleic acid nanostructure orcomplex thereof may comprise one or more faces containing one or morecoupling moieties that are configured to couple with one or morecomplementary coupling moieties on a face of a second nucleic acidnanostructure or complex thereof. A nucleic acid nanostructure orcomplex thereof may comprise a plurality of coupling moieties that areconfigured to couple with a plurality of complementary coupling moietieson a second nucleic acid nanostructure or complex thereof. In someconfigurations, a nucleic acid nanostructure or complex thereof maycomprise a plurality of coupling moieties to ensure that at least onecoupling interaction, but preferably more than one coupling interaction,is formed with a complementary nucleic acid nanostructure or complexthereof.

A nucleic acid nanostructure may comprise a plurality of coupling facesor coupling moieties that are configured to couple the nucleic acidnanostructure to a plurality of nucleic acid nanostructures. Forexample, a square- or rectangular-shaped SNAP may comprise four couplingfaces, with each coupling face configured along one of the four edgescomprising the square or rectangle. A coupling face may comprise one ormore functional groups or moieties that are configured to couple a firstnucleic acid nanostructure to a second nucleic acid nanostructure. Forexample, a coupling face or coupling moiety may comprise a plurality ofsingle-stranded nucleic acids that are configured to hybridize to aplurality of complementary single-stranded nucleic acids on a secondcoupling face or coupling moiety, thereby coupling the first couplingface to the second coupling face. In another example, a coupling facemay comprise a single streptavidin molecule that is configured to bindto a biotin molecule on a second coupling face, thereby coupling thefirst coupling face to the second coupling face. In some configurations,the coupling of a first nucleic acid nanostructure to a second nucleicacid nanostructure may comprise an intermediary coupling group thatmediates the coupling of the first nucleic acid nanostructure to thesecond nucleic acid nanostructure. For example, a plurality of SNAPs maybe configured to only display streptavidin molecules on one or morecoupling faces such that a first SNAP cannot directly bind to a secondSNAP. An intermediary coupling group comprising only surface-displayedbiotin may permit the coupling of the first SNAP to the second SNAP. Anintermediary coupling group may comprise a nucleic acid nanostructure ora non-nucleic acid particle or molecule (e.g., an organic or inorganicnanoparticle). The coupling of a first nucleic acid nanostructure to asecond nucleic acid nanostructure may be reversible (e.g., nucleic acidhybridization) or irreversible (e.g., a click reaction).

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, maycomprise one or more sites that permit controlled degradation of thenucleic acid nanostructure. A nucleic acid nanostructure may compriseone or more photocleavable linkers. Photocleavable linkers may belocated within any portion of the nucleic acid nanostructure, includingthe scaffold strand and any oligonucleotide of a plurality ofoligonucleotides that may be coupled within a nucleic acidnanostructure. In some cases, a nucleic acid nanostructure may comprisea plurality of photocleavable linkers. Photocleavable linkers may belocated within a nucleic acid nanostructure to permit controlleddegradation of the nucleic acid nanostructure, for example forprogrammed removal of the SNAP, or programmed release of the SNAP andanalyte from a surface. For nucleic acid nanostructure compositionscomprising a multifunctional moiety that is hybridized to a portion ofthe nucleic acid nanostructure, the multifunctional moiety may comprisea photocleavable linker. In some configurations, the multifunctionalmoiety may comprise no photocleavable linkers. A photocleavable linkermay be included in a multifunctional moiety to permit programmablerelease of the analyte from a nucleic acid nanostructure or a solidsupport to which the analyte is coupled. A photocleavable linker mayinclude any suitable photocleavable linker, such as nitrobenzyl,carbonyl, or benzyl-based photocleavable linkers. A photocleavablelinker may be configured to cleave under a particular wavelength, orwithin a particular frequency range, such as far infrared, nearinfrared, visible, near ultraviolet, far ultraviolet, or a combinationthereof. A photocleavable linker may be selected because it has a peakscission wavelength that does not interfere with other biological orchemical processes, such as the absorbance or emission wavelength of afluorophore. A nucleic acid nanostructure (e.g., a SNAP) may compriseone or more degradation sites that are substrates for enzymaticdegradation, for example by restriction enzymes, proteases, kinases, orother suitable enzymes. A nucleic acid nanostructure may incorporatemoieties that are substrates for enzymatic degradation, such as uracilnucleotides that are degraded by Uracil DNA glycosylase and endonucleaseVIII (sold commercially as USER® Enzyme by New England Biolabs, BeverleyMass.), 8-oxoguanine nucleotides that are degraded by DNA glycosylaseOGG1, or peptides that are degraded by proteases. For nucleic acidnanostructure compositions comprising a multifunctional moiety that ishybridized to a portion of the nucleic acid nanostructure, themultifunctional moiety may comprise a degradation site that is a targetfor enzymatic degradation. In some configurations, the multifunctionalmoiety may comprise no degradation sites that are targets for enzymaticdegradation.

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, maycomprise one or more sites or groups that are incorporated into anucleic acid nanostructure to promote stability of the nucleic acidnanostructure. A nucleic acid nanostructure (e.g., a SNAP) may comprisemodified or non-natural nucleotides (e.g., PNAs, locked nucleic acids,etc.) that are resistant to degradation via endonucleases or otherenzymes. A nucleic acid nanostructure may comprise one or morecross-linking groups that couple nucleic acid nanostructure componentsto each other (e.g., an oligonucleotide to a scaffold strand) and/or oneor more cross-linking groups that couple a nucleic acid nanostructure toanother entity (e.g., a solid support, a second nucleic acidnanostructure, etc.).

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, maycomprise one or more linkers. A linker may comprise a molecular chain ormoiety that links two portions of an oligonucleotide, including forexample, any nucleic acid components of a nucleic acid nanostructure,such as a scaffold strand, an oligonucleotide that is hybridized to ascaffold strand, or a multifunctional oligonucleotide that is hybridizedto a nucleic acid nanostructure. A linker may comprise a rigid linker ora flexible linker. A linker may comprise a polymeric moiety, such as apolyethylene glycol (PEG), a polyethylene oxide (PEO) moiety, or apolynucleotide. A linker may introduce a desired chemical property, suchas hydrophobicity, hydrophilicity, polarity, or electrical charge. Alinker may include a moiety that is configured to link one or moreadditional moieties or molecules together, such as multiplemultifunctional moieties. A linker may include one or more modifiednucleotides, such as PNAs, LNAs, and/or nucleotides modified withfunctional groups configured to perform a click-type reaction. FIG.3A-3D depicts a method of coupling an analyte to a solid supportutilizing a multifunctional moiety comprising a linking group. As shownin FIG. 3A, a SNAP 300 that is coupled to an analyte 310 by a polyvalentlinker 320 is contacted with a solid support 330 comprising a pluralityof surface-linked coupling moieties 335. The polyvalent linker iscoupled to four arms of a multifunctional moiety (321, 322, 323, 324)that are hybridized to the SNAP and comprise functional groups 325 thatare configured to couple to surface-linked coupling moieties 335. FIG.3B depicts a close-up view of the polyvalent linker 320 comprising fivefunctional groups, R₁, R₂, R₃, R₄, and R₅, respectively. Functionalgroups R₁, R₂, R₃, and R₄ are coupled to the four arms of themultifunctional moiety 321, 322, 323, and 324, respectively. Functionalgroup Rs is coupled to the analyte 310. FIG. 3C depicts the coupling ofthe SNAP 300 and analyte 310 to the solid support 330 by the coupling ofthe functional groups 325 to the surface-linked coupling moieties 335.FIG. 3D depicts the composition after the SNAP 300 structure has beendegraded, thereby leaving the analyte 310 coupled to the solid support330 by the four arms of the multifunctional moiety (321, 322, 323, 324).Such a configuration may have the advantage of increasing the chemicalstability of the coupling of the analyte as the multiple couplingmultifunctional moieties provide redundancy against decoupling of anysingle strand. The configuration may also be advantageous becausemultiple coupling multifunctional moieties may stabilize the spatialposition of the analyte where only a single coupling multifunctionalmoiety may have more translational freedom.

A nucleic acid nanostructure (e.g., a SNAP), as set forth herein, maycomprise one or more cross-linking groups. Cross-linking groups mayinclude chemical, enzymatic, and photochemical cross-linking groups. Across-linking group may stabilize or prevent the dissociation of one ormore nucleic acid structures in a nucleic acid nanostructure. Anoligonucleotide of a plurality of oligonucleotides may be cross-linkedto a scaffold strand of a nucleic acid nanostructure. A firstoligonucleotide of a plurality of oligonucleotides may be cross-linkedto a second oligonucleotide of the plurality of oligonucleotides in anucleic acid nanostructure. An oligonucleotide comprising an importantstructural feature, such as a utility moiety (e.g., a display moiety, acapture moiety) may be cross-linked to a nucleic acid nanostructure toenhance stability or prevent dissociation of the oligonucleotide. Amultifunctional moiety comprising two or more utility moieties (e.g.,display moiety and capture moiety) may comprise one or morecross-linking groups to a nucleic acid nanostructure.

A nucleic acid nanostructure (e.g., a SNAP) may comprise portions thatare fully structured and/or portions that are partially structured. Afully structured portion of a nucleic acid nanostructure may beidentified as a region of a nucleic acid nanostructure that maintainsprimary, secondary, and tertiary structure during the course of use. Apartially-structured portion of a nucleic acid nanostructure may beidentified as a region of a nucleic acid nanostructure that comprises aprimary structure but does not maintain a particular secondary and/ortertiary structure during the course of use. In some configurations, apartially-structured portion of a nucleic acid nanostructure maycomprise a single-stranded nucleic acid. A single-stranded nucleic acidmay be located between regions of double-stranded nucleic acid, or maycomprise a pendant or terminal strand of nucleic acid. A single-strandednucleic acid may have a particular length, such as, for example, atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than 50nucleotides. Alternatively or additionally, a single-stranded nucleicacid may have a length of no more than about 50, 45, 40, 35, 30, 25, 20,15, 10, 5, or less than 5 nucleotides. In some configurations, apartially-structured portion of a nucleic acid nanostructure maycomprise a non-nucleic acid moiety, molecular group or chain, such as aPEG or polymer chain. In some configurations, a partially-structuredportion of a nucleic acid nanostructure may comprise an amorphousstructure, such as a globular structure (e.g., a nanoball, a dendrimer,etc.). FIG. 37A depicts a SNAP 3710 with partially-structured regions3730 (e.g., single-stranded nucleic acids, polymers, dendrimers, etc.).The SNAP 3710 is coupled to an analyte 3720. The partially-structuredregions 3730 may be located on multiple SNAP faces (e.g., a captureface, a display face). Partially-structured regions 3730 may provide oneor more functionalities to the SNAP 3710 such as, for example,increasing binding strength to targeted binding surfaces, decreasingbinding strength to non-targeted surfaces, and prevent non-specificbinding of other molecules to a SNAP face or a coupled analyte.

Multifunctional Moieties: In an aspect, described herein is acomposition comprising a nucleic acid nanostructure (e.g., a SNAP) and amultifunctional moiety, where the multifunctional moiety may beconfigured to be coupled to the nucleic acid nanostructure, and wherethe multifunctional moiety may be configured to form two or moreadditional interactions. In some configurations, the multifunctionalmoiety may be configured to be coupled to the nucleic acidnanostructure, and may continuously couple a surface to an analyte. Acontinuous coupling of the surface to the analyte may comprise acoupling where the surface is directly coupled to the analyte by themultifunctional moiety, without any other intervening groups ormoieties. For example, if a SNAP was coupled to a surface by amultifunctional moiety and an analyte was coupled to the SNAP but notcoupled to the multifunctional moiety, the analyte would not becontinuously coupled to the surface by the multifunctional moiety. Themultifunctional moiety may comprise a first functional group and asecond functional group. In some configurations, the first functionalgroup may be coupled to, or configured to couple to, the surface, andthe second functional group may be coupled to, or configured to coupleto, the analyte. In some configurations, the multifunctional moiety maybe coupled to, or configured to be coupled to, a nucleic acidnanostructure, and may form two or more coupling interactions with asurface. A multifunctional moiety may comprise a display moiety and asurface-interacting moiety.

A multifunctional moiety, as set forth herein, may comprise a pluralityof functional groups. A multifunctional moiety may comprise at leastabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more than 20 functional groups. Alternatively or additionally, amultifunctional moiety may comprise no more than about 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less than 3functional groups.

A multifunctional moiety, as set forth herein, may comprise one or moremolecular chains. A molecular chain may comprise a multimeric compoundsuch as an oligonucleotide or a polymer chain (e.g., polyethylene,polypropylene, polyethylene glycol, polyethylene oxide, etc.). In otherconfigurations, a multifunctional moiety may comprise no nucleic acids.In some configurations a multifunctional moiety may comprise a pluralityof molecular chains. A multifunctional moiety may comprise at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 molecular chains.Alternatively or additionally, a multifunctional moiety may comprise nomore than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 molecularchains. Two or more molecular chains of a multifunctional moiety may bejoined, coupled, or linked by a linking moiety. FIGS. 7A-7B depictexemplary configurations of linking moieties. FIG. 7A depicts theformation of a multifunctional moiety comprising an alkyl linkingmoiety. The linking moiety comprises an alkyl linking group 710 thatcomprises four reactive functional groups, including 3 methyltetrazine(mTz) groups 720 and 1 dibenzocyclooctene (DBCO) group 730. The linkingmoiety may be contacted with a molecular chain 740 comprising an azidefunctional group, thereby linking the azide-functionalized molecularchain 740 to the DBCO group 730 by an azide-DBCO click reaction. Thelinking moiety may also be contacted with molecular chains 750comprising transcyclooctyne (TCO) functional groups, thereby linking theTCO-functionalized molecular chains 750 to the mTz functional groups 720by an mTz-TCO click reaction. FIG. 7B depicts a multifunctional moietycomprising a group of modified nucleotides in a longer oligonucleotidemolecular chain. The linking moiety comprising the modified nucleotidesis shown in the dashed box. The linking moiety comprises four modifiedthymine nucleotides, including two mTz-functionalized thymines 760 andtwo DBCO-functionalized thymines 770. The multifunctional moiety may becontacted with azide-functionalized molecular chains 740 and/orTCO-functionalized molecular chains 750 to couple one or more molecularchains by click reactions.

A multifunctional moiety may be configured to couple to a nucleic acidnanostructure (e.g., a SNAP). A coupling of a nucleic acid nanostructuremay depend upon how the nucleic acid nanostructure is to be utilized.For example, in some configurations, a multifunctional moiety mayfacilitate positioning and coupling a SNAP on a surface. In otherconfigurations, a SNAP may facilitate positioning and coupling amultifunctional moiety to the surface. FIGS. 8A-8D depict variousconfigurations of multifunctional moieties coupled to SNAPs. FIG. 8Ashows a multifunctional moiety 810 with functional groups R₁ and R₂comprising an oligonucleotide that couples to a SNAP 800 to form aregion of hybridized nucleic acids 830. The functional groups R₁ and R₂are displayed through a top face (e.g., a display face) and a bottomface (e.g., a capture face), respectively. FIG. 8B shows amultifunctional moiety 810 with functional groups R₁ and R₂ comprisingan oligonucleotide that couples to a SNAP 800 to form a region ofhybridized nucleic acids 830. The functional groups R₁ and R₂ aredisplayed on a bottom face (e.g., a capture face). FIG. 8C depicts amultifunctional moiety 840 with functional groups R₁ and R₂ comprising amolecular chain (e.g., a polymer, an oligonucleotide) that couples to aSNAP 800 by a functional group or moiety 850 that couples to acomplementary functional group or moiety 860 in the SNAP 800 (e.g., by aclick reaction, by nucleic acid hybridization). The functional groups R₁and R₂ are displayed through a top face (e.g., a display face) and abottom face (e.g., a capture face), respectively. FIG. 8D depicts amultifunctional moiety 840 with functional groups R₁ and R₂ comprising amolecular chain (e.g., a polymer, an oligonucleotide) that couples to aSNAP 800 by a functional group or moiety 850 that couples to acomplementary functional group or moiety 860 on an external face of theSNAP 800 (e.g., by a click reaction, by nucleic acid hybridization). Themultifunctional moiety 810 is coupled to the SNAP 800 but is configuredto be completely external to the SNAP 800 structure.

In some configurations, a nucleic acid nanostructure composition (e.g.,a SNAP composition) may comprise a nucleic acid nanostructure and amultifunctional moiety that is configured to be coupled to the nucleicacid nanostructure. In other configurations, a nucleic acidnanostructure composition may comprise a multifunctional moiety that iscoupled to the nucleic acid nanostructure. For example, a SNAPcomposition may comprise a fluidic medium that, in a firstconfiguration, contains a plurality of partially-formed SNAPs contactedwith a plurality of multifunctional moieties, and in a secondconfiguration, a plurality of fully-formed SNAPs, in which amultifunctional moiety is coupled to each SNAP. In some configurations,a nucleic acid nanostructure composition may further comprise an analytethat is configured to be coupled to the multifunctional moiety. Forexample, a SNAP composition may comprise a fluidic medium comprising aplurality of SNAPs containing multifunctional moieties and a pluralityof analytes that are configured to be coupled to the multifunctionalmoieties. In some configurations, a nucleic acid nanostructurecomposition may further comprise an analyte that is coupled to themultifunctional moiety. For example, a SNAP composition may comprise aplurality of partially formed SNAPs that are contacted with a pluralityof multifunctional moieties, in which each multifunctional moiety iscoupled to an analyte. In another example, a SNAP composition maycomprise a plurality of SNAPs containing multifunctional moieties, inwhich each multifunctional moiety is coupled to an analyte. In someconfigurations, a nucleic acid nanostructure composition may furthercomprise a surface that is configured to be coupled to themultifunctional moiety. For example, a SNAP composition may comprise asolid support comprising a plurality of surface-linked moieties, inwhich the solid support is contacted with a plurality of SNAP containingmultifunctional moieties, in which each multifunctional moiety comprisesa surface-interacting moiety that is configured to couple to asurface-linked moiety. In some configurations, a nucleic acidnanostructure composition may further comprise a surface that is coupledto the multifunctional moiety. For example, a SNAP composition maycomprise a solid support comprising a plurality of surface-linkedmoieties, in which one or more surface-linked moieties are coupled tosurface-interacting moieties of a plurality of SNAPs containingmultifunctional moieties, and in which the solid support is contactedwith a fluidic medium comprising a plurality of analytes, in which eachanalyte is configured to couple to a display moiety of a multifunctionalmoiety. The skilled person will readily recognize numerous variations ofnucleic acid nanostructure compositions based upon the ordering withwhich different components (e.g., SNAPs, multifunctional moieties,analytes, solid supports, etc.) are introduced into a system, as setforth herein.

In some configurations, provided herein are compositions comprising anucleic acid nanostructure (e.g., a SNAP) comprising a display moietythat is configured to couple to an analyte and a capture moiety that isconfigured to couple with a surface, and a multifunctional moietycomprising a first functional group and a second functional group wherethe multifunctional moiety is hybridized to a nanostructure moiety, andwhere the display moiety comprises the first functional group and thecapture moiety comprises the second functional group. Such nucleic acidnanostructures may be configured to utilize the first functional groupto couple to an analyte and to utilize the second functional group tocouple to a surface or interface. The nanostructure moiety can beconfigured to occupy a given area of the surface to prevent othernucleic acid nanostructures from occupying the same area. This canoccur, for example, due to steric exclusion, charge repulsion or othermechanisms. Such configurations may provide surprising advantages, suchas a linking connection between the analyte and the surface by themultifunctional moiety, and preventing more than one analyte fromoccupying the given area of the surface due to the presence of thenanostructure moiety. The nanostructure moiety can be removed (e.g.degraded), intentionally or unintentionally, such that the analyte mayremain coupled to the surface. Accordingly, a nanostructure moiety canbeneficially inhibit interaction of an analyte with other analytes,reagents or objects during surface deposition, and then thenanostructure moiety can be removed to facilitate interaction of theanalyte with other analytes, reagents or objects that are useful foron-surface detection or on-surface manipulation of the analyte.

FIGS. 9A-9F depict a method of coupling an analyte to a surfaceutilizing a SNAP with a multifunctional oligonucleotide. FIG. 9A depictsa schematic of a SNAP 910 comprising an oligonucleotide 940 with a firstterminal functional group 920 comprising dibenzocyclooctyne (DBCO) and asecond terminal functional group 930 comprising methyltetrazine (mTz).The oligonucleotide 940 is configured to hybridize to a portion of theSNAP such that it forms a localized region of secondary or tertiarystructure 945 (e.g., a double helix), thereby stabilizing theoligonucleotide 940 within the SNAP structure 910. The SNAP 910 iscontacted with a solid support 950 comprising non-reactive regions 952,and region comprising a reactive third functional group 955 comprisingan azide moiety that is configured to react with the first terminalfunctional group 920. As shown in FIG. 9B, the first terminal functionalgroup 920 may react with the third functional group 955 to form acovalent bond that couples the SNAP 910 to the solid support 950 in thevicinity of where the third functional group 955 is coupled to the solidsupport 950. As shown in FIG. 9C, the coupled SNAP may be contacted withan analyte 960 comprising a fourth functional group 970 comprisingtranscyclooctene that is configured to react with the second terminalfunctional group 930. As shown in FIG. 9D, the second terminalfunctional group 930 may react with the fourth functional group 970 toform a covalent bond that couples the analyte 960 to the solid support950. It will be understood that functional groups 920, 955, 930 and 970are exemplary and can be replaced with other coupling moieties such asthose set forth herein or known in the art. As shown in FIG. 9E, theSNAP-analyte composition may be subjected to a degrading phenomena, suchas a light source 980, that disrupts the structure of the SNAP 910,thereby degrading the SNAP 910. Degradation can be carried out usingother means such as endonuclease digestion of one or more nucleic acidstrands in the SNAP, thermal or chemical denaturation of nucleic acidstrand interaction, or chemical lysis of a scissile linkage in the SNAP.As shown in FIG. 9F, after degradation of the SNAP 910, the analyte 960may remain coupled to the solid support 950 by the oligonucleotide 940.

A nucleic acid nanostructure (e.g., a SNAP) comprising a multifunctionalmoiety, such as the configurations depicted in FIGS. 9A-9F, may beconfigured to form a hybridization region with the multifunctionalmoiety consisting of a plurality of nucleic acid base pairs. In someconfigurations, a multifunctional moiety may form a hybridization regionwith a nucleic acid nanostructure comprising at least about 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 125, 150, 200, or more than 200nucleotides. Alternatively or additionally, a multifunctional moiety mayform a hybridization region with a nucleic acid nanostructure comprisingno more than about 200, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20,10, or less than about 10 nucleotides. A hybridization region formedbetween a nucleic acid nanostructure and a multifunctional moiety may becharacterized by a particular number of helical revolutions formed(where a single revolution usually comprises between 10 and 11 basepairs). In some configurations, a multifunctional moiety may form ahybridization region comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 helicalrevolutions. Alternatively or additionally, a multifunctional moiety mayform a hybridization region comprising no more than 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less than 1 helicalrevolution.

A nucleic acid nanostructure (e.g., a SNAP) may comprise a plurality oftertiary structures that collectively form quaternary or other higherorder structures in the nucleic acid nanostructure. Particular tertiarystructures may comprise moieties or structures that belong to aparticular face of the nucleic acid nanostructure. A nucleic acidnanostructure may comprise a plurality of tertiary structures, where adisplay face comprises a first tertiary structure of the plurality oftertiary structures, and a capture face comprises a second tertiarystructure of the plurality of tertiary structures. In someconfigurations, the first tertiary structure may be the same as thesecond tertiary structure. In other configurations, the first tertiarystructure is different from the second tertiary structure. In nucleicacid nanostructure configurations comprising a multifunctional moietywith a first functional group and a second functional group, themultifunctional moiety may be hybridized to the nucleic acidnanostructure, thereby forming a portion of the first tertiary structureor a portion of the second tertiary structure. In other configurations,the multifunctional moiety may be hybridized to a nucleic acidnanostructure, thereby forming a portion of both the first tertiarystructure and a portion of the second tertiary structure.

A nucleic acid nanostructure (e.g., a SNAP) comprising a firstmultifunctional moiety may further comprise a second multifunctionalmoiety that comprises a third functional group and a fourth functionalgroup. In some configurations, a utility moiety (e.g., a display moiety)may comprise a third functional group and a second utility moiety (e.g.,a capture moiety) may comprise a fourth functional group. In someconfigurations, a third or fourth functional group may be configured tocouple to a surface. In some specific configurations, a third or fourthfunctional group may be coupled to a surface. In some configurations, athird or fourth functional group may be configured to couple to a secondanalyte. In some specific configurations, a third or fourth functionalgroup may be coupled to a second analyte. In some configurations, athird or fourth functional group may be configured to be coupled to ananalyte to which a first multifunctional moiety is coupled. In somespecific configurations, a third or fourth functional group may becoupled to an analyte to which a first multifunctional moiety iscoupled.

FIG. 10A-10D illustrate a method of coupling a SNAP comprising twomultifunctional moieties to a surface. FIG. 10A shows a SNAP 1000comprising a first multifunctional moiety 1010 that is coupled to ananalyte 1020 and comprises a first functional group 1015. The SNAP 1000also comprises a second multifunctional moiety 1030 that is coupled to autility moiety 1040 and comprises a second functional group 1035. TheSNAP 1000 may comprise a capture face comprising a capture moietycontaining the first functional group and the second functional group.The SNAP 1000 may be contacted with a solid support 1050 comprising aplurality of functional groups or moieties including surface linkednon-coupling groups 1060 and surface-linked coupling groups 1065 thatare configured to couple to a capture moiety or a plurality of capturemoieties. As shown in FIG. 10B, the first functional group and/or thesecond functional group may couple to a surface linked coupling group1065, thereby coupling the SNAP 1010 to the solid support 1050 by atleast one of the two functional groups comprising the capture moiety. Asshown in FIG. 10C, the SNAP 1000 coupled to the solid support 1050 maybe exposed to a degrading phenomenon, such as a light source 1070, thatcauses degradation of the SNAP 1000 structure. Degradation can becarried out using other means such as endonuclease digestion of one ormore nucleic acid strands in the SNAP, heat, pH change, chemical lysisof a scissile linkage in the SNAP, or any other suitable method ofdegradation. As shown in FIG. 10D, after degradation of the SNAPstructure, the first multifunctional moiety 1010 that is coupled to theanalyte 1020 and the second multifunctional moiety 1030 that is coupledto a utility moiety 1040 are co-localized on the solid support 1050.

A multifunctional moiety that is hybridized to a nucleic acidnanostructure structure (e.g., a SNAP) may be configured to couple thenucleic acid nanostructure or a moiety thereof to a surface. In someconfigurations, a surface may comprise a surface functional group thatis configured to couple to a functional group contained on amultifunctional moiety. In some configurations, a surface functionalgroup may comprise a functional group that is configured to form acovalent bond with a functional group contained on a multifunctionalmoiety. In some specific configurations, a surface functional group anda functional group contained on a multifunctional moiety may form acovalent bond, for example by a click-type reaction, a substitutionreaction, an elimination reaction, or any other suitable bondingchemistry.

A nucleic acid nanostructure (e.g., a SNAP) comprising a multifunctionalmoiety may be formed before or after coupling the nucleic acidnanostructure with a surface. FIG. 11A-11D depict a method ofhybridizing a multifunctional moiety to a SNAP after the SNAP has beencoupled to a surface. FIG. 11A shows a SNAP 1110 that is contacted witha surface 1100, thereby permitting the SNAP to couple to the surface,for example by an electrostatic, magnetic, or covalent interaction. FIG.11B shows a contacting of a multifunctional moiety 1120 that is coupledto an analyte 1130 with the SNAP 1110 coupled to the surface 1100. Asshown in FIG. 11C, the multifunctional moiety 1120 hybridizes to theSNAP 1110, forming a region of tertiary structure 1150. Themultifunctional moiety 1120 may further couple to the surface 1100. FIG.11D depicts a continuous linkage of the analyte 1130 to the surface 1100after the SNAP 1110 is optionally removed.

Partially-Compacted Nucleic Acids: A nucleic acid that is useful for theformation of an array of analytes may comprise a structure that has oneor more characteristics of: i) coupling an analyte at a tunable and/orcontrollable location on a face of the nucleic acid, ii) inhibitingunwanted coupling of analytes or other moieties at portions of thenucleic acid not intended for coupling, iii) comprising a structure or aface that is configured to form a specific binding interaction with asolid support or a surface thereof, iv) comprising a structure or a facethat is configured to form a specific binding interaction with a solidsupport or a surface thereof that is more likely to occur than anon-specific binding interaction between an analyte coupled to thenucleic acid and the solid support or surface thereof, v) comprising astructure or a face that is configured to inhibit contact between ananalyte coupled to the nucleic acid and a solid support or a surfacethereof, vi) inhibiting unwanted binding interactions (e.g.,aggregation, co-localization, etc.) with other nucleic acids or analytescoupled thereto.

A useful configuration of a nucleic acid, such as a nucleic acidnanostructure, may comprise a nucleic acid comprising a compactedstructure and a pervious structure. A compacted structure of a nucleicacid may provide spatial and orientational tunability for moietiescoupled to or emerging from a structure of a nucleic acid. For example,a nucleic acid origami comprising a compacted structure may be designedto orient a display moiety at substantially a 180° orientation from oneor more capture moieties, thereby increasing likelihood that the nucleicacid origami is coupled to a solid support by the one or more capturemoieties rather and not coupled by an analyte coupled to the displaymoiety. Tunability of a compacted structure may arise from severalaspects of a nucleic acid structure, including a plurality of tertiarystructures that provide substantially 360° of rotational freedom for theorientation of moieties coupled to a nucleic acid, and one or morelinking strands that couple tertiary structures within a nucleic acidstructure, thereby providing a degree of rigidity to the nucleic acidstructure and fixing the separation distance and orientation of tertiarystructures with respect to each other in the nucleic acid structure. Apervious structure of a nucleic acid may provide additional chemicaland/or physical properties to a nucleic acid that facilitate wantedinteractions with other entities (e.g., analytes, unbound moieties,reagents, other nucleic acids, solid supports, fluidic media, etc.) orinhibit unwanted interactions with other entities. For example, anucleic acid may comprise a plurality of pendant single-stranded nucleicacid moieties comprising homopolymer repeats (e.g., poly-T repeats,poly-A repeats, poly-C repeats, poly-G repeats), in which the pendantsingle-stranded nucleic acid moieties are configured to inhibitco-localization of two or more nucleic acids on a solid support (e.g.,at the same address in an array of addresses on a solid support). Bycoupling a pervious structure to a tunable compacted structure of anucleic acid, the location and orientation of the pervious structure canbe controlled to produce more specific and localized interactionsbetween the nucleic acid and other entities.

A nucleic acid nanostructure, as set forth herein, may comprise at leastone compacted region or structure. A compacted region of a nucleic acidnanostructure may refer to a region or structure with an averagecharacteristic closer to an average characteristic for a multi-strandednucleic acid (e.g., double-stranded DNA, triple-stranded DNA, etc.)relative to a single-stranded nucleic acid. A nucleic acidnanostructure, as set forth herein, may comprise at least one perviousregion or structure. A pervious region of a nucleic acid nanostructuremay refer to a region or structure with an average characteristic closerto an average characteristic for a single-stranded nucleic acid relativeto a multi-stranded nucleic acid. A nucleic acid nanostructure, as setforth herein, need not comprise a pervious region or structure. Acompacted region or structure of a nucleic acid nanostructure maycomprise one or more characteristics of: i) comprising a scaffoldstrand, ii) comprising a plurality of nucleic acids coupled to ascaffold strand, in which at least 50%, and optionally at least 60%,70%, 75%, 80%, 85%, 90%, or 95% of nucleotides of the scaffold strandare base-pair hybridized to nucleotides of the plurality of nucleicacids, iii) comprising a plurality of coupled nucleic acids, in which atleast 50%, and optionally at least 60%, 70%, 75%, 80%, 85%, 90%, or 95%of nucleotides of the plurality of nucleic acids are base-pairhybridized to other nucleotides of the plurality of nucleic acids, iv)comprising a plurality of secondary and/or tertiary nucleic acidstructures, in which a position, orientation, and/or motion of a firstsecondary and/or tertiary nucleic acid structure relative to a secondsecondary and/or tertiary nucleic acid structure is constrained, v)comprising a first helical nucleic acid structure and a second helicalnucleic acid structure, in which the first helical nucleic acidstructure and the second helical nucleic acid structure are linked by asingle-stranded nucleic acid, in which the first helical nucleic acidstructure and the second helical nucleic acid structure each comprise ahelical axis of symmetry parallel oriented in a 3′ to 5′ directionrelative to the single-stranded nucleic acid, and in which anorientation of the helical axis of symmetry of the first helical nucleicacid structure relative to the helical axis of symmetry of the secondhelical nucleic acid structure has an angle between about 90° and 180°,vi) comprising a single-stranded nucleic acid that constrains aposition, orientation, and/or motion of a first secondary and/ortertiary nucleic acid structure relative to a second secondary and/ortertiary nucleic acid structure; vii) comprising a moiety (e.g., apolypeptide, a polysaccharide, a nanoparticle, etc.) that constrains aposition, orientation, and/or motion of a first secondary and/ortertiary nucleic acid structure relative to a second secondary and/ortertiary nucleic acid structure; viii) comprising a volume that encloseseach nucleotide of the compacted region or structure, in which acharacteristic dimension of the volume (e.g., a length, a depth, adiameter, etc.) does not vary by more than 10%, and optionally by nomore than 5% or 1% due to intermolecular or extramolecular motion (e.g.,Brownian motion, fluidic shear, electromagnetic forces, etc.), or due tointramolecular motion (e.g., translation, vibration, bending, rotation,etc.), ix) comprising a first nucleotide with a first tunable locationand a second nucleotide with a second tunable location, in which thefirst tunable location comprises a distance from and orientationrelative to the second tunable location, x) comprising a firstnucleotide with a first tunable location and a second nucleotide with asecond tunable location, in which the first tunable location comprises adistance from or an orientation relative to the second tunable locationthat varies by no more than 10%, xi) comprising a volume that encloseseach nucleotide of the compacted region or structure, in which acharacteristic dimension of the volume (e.g., a length, a depth, adiameter, etc.) does not vary by more than 10%, and optionally no morethan 5%, or 1%, when the nucleic acid nanostructure comprising thecompacted region or structure forms a binding interaction with amolecule, moiety, structure, or solid support, xii) comprising atwo-dimensional projection of an area of the compacted region orstructure that surrounds each nucleotide of the compacted region orstructure, in which the two-dimensional projection does not vary by morethan 10%, and optionally no more than 5%, or 1%, when the nucleic acidnanostructure comprising the compacted region or structure forms abinding interaction with a molecule, moiety, structure, or solidsupport, xiii) comprising a plurality of single-stranded nucleic acids,in which each single-stranded nucleic acid is less than about 20nucleotides in length, and optionally no more than about 15, 10, or 5nucleotides in length, xiv) comprising a first tertiary structure and asecond tertiary structure, in which the second tertiary structure isadjacent to the first tertiary structure, and in which an averageseparation distance between the first tertiary structure and thesecondary structure is no more than about 20 nanometers (nm), andoptionally no more than about 10 nm or 5 nm as measured by an averageseparation distance between an axis of symmetry for the first tertiarystructure and an axis of symmetry for the second tertiary structure, xv)comprising a first tertiary structure and a second tertiary structure,in which the second tertiary structure is adjacent to the first tertiarystructure, in which the first tertiary structure and the second tertiarystructure each comprise a common nucleic acid, and optionally two commonnucleic acids, and in which the common nucleic acid comprises a bend ofat least about 90°, in which the bend has a radius of curvature of nomore than 10 nanometers (nm), and optionally no more than 5 nm or 2.5nm, and xvi) comprising a first tertiary structure and a second tertiarystructure, in which the second tertiary structure is adjacent to thefirst tertiary structure, in which the first tertiary structure and thesecond tertiary structure each comprise a common nucleic acid, andoptionally two common nucleic acids, in which the common nucleic acidcomprises a bend of at least about 90°, in which the bend has a radiusof curvature of no more than 10 nanometers (nm), and optionally no morethan 5 nm or 2.5 nm, and in which the first tertiary structure is notpositioned adjacent to the second tertiary structure by a nucleicacid-binding entity (e.g., a nucleic acid-binding protein, ananoparticle, etc.).

A nucleic acid nanostructure, as set forth herein, may comprise at leastone pervious region or structure. A pervious region or structure of anucleic acid nanostructure may comprise one or more characteristics of:i) not comprising a scaffold strand, ii) comprising one or more nucleicacids, in which each nucleic acid of the one or more nucleic acidscomprises a first nucleotide sequence that is configured to hybridize toa scaffold strand of a compacted region or structure, and a secondnucleotide sequence that is not configured to hybridize to an nucleicacid of the nucleic acid nanostructure, iii) comprising one or morenucleic acids, in which each nucleic acid of the one or more nucleicacids comprises a single-stranded nucleic acid of at least about 20nucleotides in length, and optionally at least about 25, 50, 100, 500,1000, or more than 1000 nucleotides in length, iv) comprising one ormore nucleic acids, in which each nucleic acids of the one or morenucleic acids comprises an uncoupled terminal nucleotide (e.g., a 3′terminal nucleotide, a 5′ terminal nucleotide), v) comprising aplurality of pendant moieties (e.g., single-stranded nucleic acids,partially-double-stranded nucleic acids, polymer chains, etc.), in whicheach pendant moiety comprises a position, orientation, or motion that isnot constrained by an intramolecular or intrastructure bindinginteraction (e.g., base-pair hybridization, hydrogen-bonding, van derWaals interactions, etc.), vi) comprising a plurality of pendantmoieties, in which each pendant moiety comprises a position,orientation, or motion that is constrained by a non-binding interaction(e.g., steric occlusion, electrostatic repulsion, magnetic repulsion,hydrophobic interactions, hydrophilic interactions, vii) comprising oneor more coupled nucleic acids, in which less than 50%, and optionallyless than 40%, 30%, 20%, 10%, 5%, or 1% of nucleotides of the pluralityof nucleic acids are base-pair hybridized to other nucleotides of theplurality of nucleic acids, ix) comprising one or more nucleic acids, inwhich the one or more nucleic acids comprise a first single-strandednucleic acid and a second single-stranded nucleic acid, in which thefirst single-stranded nucleic acid is not configured to hybridize to thesecond single-stranded nucleic acid, x) comprising one or more nucleicacids, in which the one or more nucleic acids comprise a single-strandednucleic acid comprising a polynucleotide repeat (e.g., poly-A, poly-C,poly-G, poly-T), optionally in which the polynucleotide repeat comprisesat least about 10 nucleotides, or at least about 20, 30, 40, 50, 100,200, 500, 1000, or more than 1000 nucleotides, xi) comprising a volumethat encloses each nucleotide of the pervious region or structure, inwhich a characteristic dimension of the volume (e.g., a length, a depth,a diameter, etc.) varies by more than 10%, and optionally by more than15% or 20% due to intermolecular or extramolecular motion (e.g.,Brownian motion, fluidic shear, electromagnetic forces, etc.), or due tointramolecular motion (e.g., translation, vibration, bending, rotation,etc.), xii) comprising a volume that encloses each nucleotide of thepervious region or structure, in which a characteristic dimension of thevolume (e.g., a length, a depth a diameter, etc.) varies by more than10%, and optionally more than 15% or 20%, when the nucleic acidnanostructure comprising the compacted region or structure forms abinding interaction with a molecule, moiety, structure, or solidsupport, xiii) comprising a two-dimensional projection of an area of thepervious region or structure that surrounds a furthest extent of thepervious region or structure when the nucleic acid nanostructure is notcoupled to a molecule, moiety, structure or location, in which thetwo-dimensional projection varies by more than 10%, and optionally nomore than 15%, or 20%, when the nucleic acid nanostructure comprisingthe pervious region or structure forms a binding interaction with themolecule, moiety, structure, or solid support, and xiv) comprising annucleic acid, in which a first nucleotide sequence of the nucleic acidis coupled to a compacted structure, in which a second nucleotidesequence of the nucleic acid is not coupled to a compacted structure,and in which a nucleotide of the second nucleotide sequence comprises alarger spatial and/or temporal variation of a standard deviation indistance to the compacted structure relative to a nucleotide of thefirst nucleotide sequence.

In an aspect, provided herein is a nucleic acid nanostructure,comprising at least 10 coupled nucleic acids, in which the nucleic acidnanostructure comprises: a) a compacted region comprising high internalcomplementarity, in which the high internal complementarity comprises atleast 50% double-stranded nucleic acids and at least 1% single-strandednucleic acids, and in which the compacted region comprises a displaymoiety, in which the display moiety is coupled to, or configured tocouple to, an analyte of interest, and b) a pervious region comprisinglow internal complementarity, in which the low internal complementaritycomprises at least about 50% single-stranded nucleic acids, and in whichthe pervious region comprises a coupling moiety, in which the couplingmoiety forms, or is configured to form, a coupling interaction with asolid support.

In another aspect, provided herein is a nucleic acid nanostructure,comprising: a) a compacted structure, in which the compacted structurecomprises a scaffold strand and a first plurality of stapleoligonucleotides, in which at least 80% of nucleotides of the scaffoldstrand are hybridized to nucleotides of the first plurality of stapleoligonucleotides, in which the first plurality of stapleoligonucleotides hybridizes to the scaffold strand to form a pluralityof tertiary structures, in which the plurality of tertiary structuresincludes adjacent tertiary structures linked by a single-stranded regionof the scaffold strand, and in which the relative positions of theadjacent tertiary structures are positionally constrained, and b) apervious structure, in which the pervious structure comprises a secondplurality of staple oligonucleotides, in which the stapleoligonucleotides are coupled to the scaffold strand of the compactedstructure, in which the pervious structure comprises at least 50%single-stranded nucleic acid, and in which the pervious structure has ananisotropic three-dimensional distribution around at least a portion ofthe compacted structure.

In another aspect, provided herein is a nucleic acid nanostructure,comprising: a) a compacted structure, in which the compacted structurecomprises a scaffold strand and a first plurality of stapleoligonucleotides, in which at least 80% of nucleotides of the scaffoldstrand are hybridized to nucleotides of the first plurality of stapleoligonucleotides, in which the first plurality of stapleoligonucleotides hybridizes to the scaffold strand to form a pluralityof tertiary structures, in which the plurality of tertiary structuresincludes adjacent tertiary structures linked by a single-stranded regionof the scaffold, in which the relative positions of the adjacenttertiary structures are positionally constrained, and in which thecompacted structure comprises an effective surface area; and b) apervious structure, in which the pervious structure comprises a secondplurality of staple oligonucleotides, in which the stapleoligonucleotides are coupled to the scaffold strand of the compactedstructure, in which the pervious structure comprises at least 50%single-stranded nucleic acid, and in which (i) the effective surfacearea of the nucleic acid nanostructure is larger than the effectivesurface area of the compacted structure or (ii) the ratio of effectivesurface area to volume of the nucleic acid nanostructure is larger thanthe ratio of effective surface area to volume of the compactedstructure.

In another aspect, provided herein is a nucleic acid nanostructure,comprising a plurality of nucleic acid strands, in which each strand ofthe plurality of strands is hybridized to another strand of theplurality of strands to form a plurality of tertiary structures, and inwhich a strand of the plurality of strands comprises a first nucleotidesequence that is hybridized to a second strand of the plurality ofstrands, in which the strand of the plurality of strands furthercomprises a second nucleotide sequence of at least 100 consecutivenucleotides, and in which at least 50 nucleotides of the secondnucleotide sequence is single-stranded.

FIGS. 52A-52H illustrate various configurations of nucleic acidnanostructure comprising a compacted structure and a pervious structure.FIG. 52A depicts a cross-sectional view of a nucleic acid nanostructurecomprising a SNAP 5210 (e.g., a nucleic acid origami) coupled to ananalyte 5220 by a display moiety 5215 on a display face of the SNAP5210. The nucleic acid nanostructure further comprises a capture facethat is opposite (e.g., about 180° in orientation from) the display faceof the SNAP 5210. The capture face comprises a pervious structurecomprising a plurality of pendant moieties 5212 (e.g., single-strandednucleic acids, polymer chains, etc.) that are coupled to the captureface of the SNAP 5210, in which the pendant moieties 5212 compriseunbound termini. Depending upon the density of the plurality of pendantmoieties 5212 and the rigidness of the coupling points to the compactedstructure of the SNAP 5210, the plurality of pendant moieties mayarrange in an outwardly-fanned configuration. Volume 5230 encloses anaverage space occupied by the pervious structure comprising theplurality of pendant moieties. The pendant moieties 5212 within volume5230 have an anisotropic spatial distribution with respect to thecompacted structure of the SNAP 5210 due to the tunable positioning andorientation of the pendant moieties on the capture face of the SNAP5210. FIG. 52B illustrates a top-down view of the nucleic acidnanostructure in FIG. 52A. Line 5241 outlines the effective surface areaof the compacted structure of the SNAP 5210 and line 5240 outlines theeffective surface area of the complete nucleic acid nanostructure (i.e.including the compacted structure and the pervious structure), which isgreater than the effective surface area of the compacted structure dueto the outward fanning of the pendant moieties 5212.

FIG. 52C depicts a cross-sectional view of a nucleic acid nanostructurecomprising a SNAP 5210 (e.g., a nucleic acid origami) coupled to ananalyte 5220 by a display moiety 5215 on a display face of the SNAP5210. The nucleic acid nanostructure further comprises one or moreutility faces that are adjacent and orthogonal to (e.g., about 90° inorientation from) the display face of the SNAP 5210. Each utility facecomprises a pervious structure comprising a plurality of pendantmoieties 5212 (e.g., single-stranded nucleic acids, polymer chains,etc.) that are coupled to the utility face of the SNAP 5210. Dependingupon the density of the plurality of pendant moieties 5212, theflexibility of the pendant moieties 5212 and the rigidness of thecoupling points to the compacted structure of the SNAP 5210, theplurality of pendant moieties 5212 may arrange in an outwardly-fannedconfiguration. lines 5230 and 5231 encloses an average cross-sectionalarea of the space occupied by the pervious structure comprising theplurality of pendant moieties. Pendant moieties 5212 within the spaceindicated by lines 5230 and 5231 comprise a substantially isotropicspatial distribution with respect to the midline of the compactedstructure of the SNAP 5210 and an anisotropic spatial distributionrelative to the analyte 5220 due to the tunable positioning andorientation of the pendant moieties on the capture face of the SNAP5210. FIG. 52D illustrates a top-down view of the nucleic acidnanostructure. Line 5241 outlines the effective surface area of thecompacted structure of the SNAP 5210 and line 5240 outlines theeffective surface area of the complete nucleic acid nanostructure (i.e.including the compacted structure and the pervious structure), which isgreater than the effective surface area of the compacted structure dueto the outward direction of the pedant moieties 5212.

FIG. 52E depicts a cross-sectional view of a nucleic acid nanostructurecomprising a SNAP 5210 (e.g., a nucleic acid origami) coupled to ananalyte 5220 by a display moiety 5215 on a display face of the SNAP5210. The nucleic acid nanostructure further comprises a capture facethat is opposite (e.g., about 180° in orientation from) the display faceof the SNAP 5210. The capture face comprises a pervious structurecomprising a plurality of pendant moieties 5213 (e.g., single-strandednucleic acids, polymer chains, etc.) that are coupled to the captureface of the SNAP 5210, in which the pendant moieties 5213 have bothtermini coupled to the compacted structure of the SNAP 5210. Dependingupon the density of the plurality of pendant moieties 5213, theirflexibility and the rigidness of the coupling points to the compactedstructure of the SNAP 5210, the plurality of pendant moieties may occupya volume directly below the capture face of the SNAP 5210. Line 5230encloses an average cross-sectional area of the space occupied by thepervious structure comprising the plurality of pendant moieties 5213.Pendant moieties 5213 within the space indicated by line 5230 comprisesan anisotropic spatial distribution with respect to the compactedstructure of the SNAP 5210 due to the tunable positioning andorientation of the pendant moieties on the capture face of the SNAP5210. FIG. 52F illustrates a top-down view of the nucleic acidnanostructure. Line 5241 outlines the effective surface area of thecompacted structure of the SNAP 5210 and line 5240 outlines theeffective surface area of the complete nucleic acid nanostructure (i.e.including the compacted structure and the pervious structure), which issmaller than the effective surface area of the compacted structure ofthe SNAP 5210.

FIG. 52G depicts a cross-sectional view of a nucleic acid nanostructurecomprising a SNAP 5210 (e.g., a nucleic acid origami) coupled to ananalyte 5220 by a display moiety 5215 on a display face of the SNAP5210. The nucleic acid nanostructure further comprises a plurality ofpendant moieties 5212 (e.g., single-stranded nucleic acids, polymerchains, etc.) that are coupled to nearly all orientations of the SNAP5210 excluding a volume occupied by the analyte 5220. Depending upon thedensity of the plurality of pendant moieties 5212, their flexibility andthe rigidness of the coupling points to the compacted structure of theSNAP 5210, the plurality of pendant moieties may arrange in anoutwardly-fanned configuration. Line 5230 encloses an averagecross-section of the space occupied by the pervious structure comprisingthe plurality of pendant moieties 5212. Pendant moieties 5212 within thespace indicated by line 5230 comprises an anisotropic spatialdistribution with respect to the compacted structure of the SNAP 5210although it may be an isotropic spatial distribution excluding thevolume occupied by the analyte 5220. FIG. 52H illustrates a top-downview of the nucleic acid nanostructure. Line 5241 outlines the effectivesurface area of the compacted structure of the SNAP 5210 and line 5240outlines the effective surface area of the complete nucleic acidnanostructure (i.e. including the compacted structure and the perviousstructure), which is greater than the effective surface area of thecompacted structure due to the outward fanning of the pedant moieties5212.

FIGS. 53A-53E depict cross-sectional views of various nucleic acidnanostructure configurations, in which each nucleic acid nanostructurecomprises a pervious structure, and in which each pervious structurecomprises a plurality of pendant moieties that are configured to havediffering interactions with other entities (e.g., analytes, othernucleic acid nanostructures, solid supports, reagents, etc.). FIG. 53Adepicts a compacted structure 5310 (e.g., a SNAP) that is coupled to apervious structure comprising a plurality of pendant oligonucleotides5320, in which each pendant oligonucleotide comprises a homopolymer. Thehomopolymer of each pendant oligonucleotide 5320 may inhibit bindinginteractions with other nucleic acid nanostructures having the same orsimilar pendant oligonucleotide sequences. FIG. 53B depicts a compactedstructure 5310 (e.g., a SNAP) that is coupled to a pervious structurecomprising a plurality of pendant oligonucleotides 5321, in which eachpendant oligonucleotide comprises homopolymer sequences, and in whichsome homopolymers are interrupted by random substitutions of nucleotidesother than the nucleotide of the homopolymer sequence (e.g., a poly-Tsequence comprising randomly-substituted A, C, or G nucleotides). FIG.53C depicts a compacted structure 5310 (e.g., a SNAP) that is coupled toa pervious structure comprising a plurality of pendant oligonucleotides5320, in which each pendant oligonucleotide comprises a homopolymersequence region, and a sequence region that complements the homopolymersequence region. As shown the complementary regions can form a doublestranded region 5322 to form a loop structure. FIG. 53D depicts acompacted structure 5310 (e.g., a SNAP) that is coupled to a perviousstructure comprising a plurality of pendant oligonucleotides 5323, inwhich each pendant oligonucleotide comprises a nucleotide sequence witha degree of self-complementarity (e.g., forming a stem, loop, hairpin,or bulge structure). FIG. 53E depicts a compacted structure 5310 (e.g.,a SNAP) that is coupled to a pervious structure comprising a pluralityof pendant oligonucleotides 5324, in which each pendant oligonucleotidecomprises a second oligonucleotide 5325 that hybridizes to the pendantoligonucleotide 5324. The configurations illustrated in FIGS. 53A-53E(e.g., polynucleotide repeats, random nucleotide substitutions,self-complementarity, intermittent secondary structure) may facilitatere-arrangement of orientation of the nucleic acid nanostructure on acoupling surface, thereby facilitating positioning of the nucleic acidnanostructure in a stable configuration on the coupling surface.

FIGS. 54A-54C illustrate a schematic for methods of producing nucleicacid nanostructures in accordance with some embodiments set forth herein(e.g., nucleic acid nanostructures depicted in FIGS. 53A-53E). FIG. 54Adepicts a method of forming a nucleic acid nanostructure comprising aplurality of pendant moieties comprising polynucleotide repeats. In afirst step, a scaffold strand 5410 may be combined, optionally at anelevated temperature, with a plurality of staple oligonucleotides 5420that hybridize to the scaffold strand 5410 to form a compactedstructure, and a plurality of oligonucleotides 5421 that comprisependant nucleotide sequences 5422. After cooling the oligonucleotidemixture, a nucleic acid nanostructure is formed comprising a compactedstructure 5430 and a plurality of pendant moieties comprising thependant nucleotide sequences 5422. In a second step, the nucleic acidnanostructures are subsequently contacted with a nucleic acid extensionenzyme (e.g., terminal deoxynucleotide transferase or TdT is shown) inthe presence of a homogeneous plurality of nucleotides (e.g.deoxythymidine) to produce a plurality of pendant homopolymericpolynucleotides 5423 (e.g., poly-T repeats). Optionally, the nucleotidesprovided to the enzyme may comprise small quantities of othernucleotides to generate randomly incorporated nucleotides in thepolynucleotide repeats. FIG. 54B depicts a method of forming a nucleicacid nanostructure comprising a plurality of pendant moieties comprisinghomopolymeric polynucleotides, in which the location of each pendantmoieties is controlled. In a first step, a scaffold strand 5410 may becombined, optionally at an elevated temperature, with a plurality ofstaple oligonucleotides 5420 that hybridize to the scaffold strand 5410to form a compacted structure, and a plurality of oligonucleotides 5421that comprise pendant nucleotide sequences 5422, as well as a pluralityof oligonucleotides 5424 comprising a capping moiety 5425 (e.g., adideoxynucleotide, a terminator nucleotide, a phosphorylated nucleotide,a terminal residue to buries within the compacted structure 5430, etc.),in which the capping moiety is configured to inhibit the activity of anucleic acid extension enzyme. After cooling the oligonucleotidemixture, a nucleic acid nanostructure is formed comprising a compactedstructure 5430 and a plurality of pendant moieties comprising thependant nucleotide sequences 5422 at least some of which include thecapping moiety 5425. In a second step, the nucleic acid nanostructuresare subsequently contacted with a nucleic acid extension enzyme (e.g.,terminal deoxynucleotide transferase or TdT) in the presence of ahomogeneous plurality of nucleotides (e.g. deoxythymidine) to produce aplurality of pendant polynucleotide repeats 5423 (e.g., poly-T repeats)at any pendant oligonucleotide that does not comprise a capping moiety5425. FIG. 54C depicts a method of forming a nucleic acid nanostructurecomprising a plurality of pendant moieties comprising homopolymericsequence, in which the homopolymeric sequence is interrupted by anintermediate nucleotide sequence. Nucleic acid nanostructures are formedaccording to the first step described in FIG. 54A. Optionally, thesecond step depicted in FIG. 54A may be performed to add a homopolymericsequence to each pendant moiety. In a second step, a polymeraseextension reaction is performed whereby pendant primers 5422 hybridizeto template nucleic acids that contain a complement of an intermediatenucleotide sequence 5426. The polymerase extension reaction will producependant oligonucleotides including primer sequence 5422 and intermediatenucleotide sequence 5426. In a third step, the enzymatic extension stepof FIG. 54A is performed using TdT and nucleotides to form nucleic acidnanostructures with a plurality of pendant moieties, in which eachpendant moiety comprises an intermediate nucleotide sequence 5426flanked by pendant primer sequence 5422 and homopolymer sequence 5427.

A nucleic acid nanostructure or a component structure thereof, as setforth herein, may comprise regions of internal complementarity. Internalcomplementarity may refer to the extent of double-stranded nucleic acidwithin a nucleic acid nanostructure or a component structure thereof.Internal complementarity may be quantified as a percentage ofnucleotides with a base pair complement in a formed nucleic acidnanostructure or a component structure thereof (e.g., a compactedstructure, a pervious structure). Extent of internal complementarity maybe calculated with respect to total nucleotide content. For example, anucleic acid nanostructure may comprise 10000 total nucleotides amongstat least 200 oligonucleotides that form the nucleic acid nanostructure,in which 8500 nucleotides have a base pair complement in a doublestranded region, giving the nucleic acid nanostructure 85% internalcomplementarity. An extent of internal complementarity may be calculatedwith respect to a single nucleic acid (e.g., a scaffold strand) or asubset of oligonucleotides comprising a nucleic acid nanostructure or acomponent structure thereof. For example, a compacted structure of anucleic acid nanostructure may comprise a scaffold strand of at least7000 nucleotides, in which at least 90% of the nucleotides of thescaffold strand have a base-pair complement in a double stranded region.In another example, a pervious structure of a nucleic acid nanostructuremay comprise a plurality of pendant moieties, in which each pendantmoiety comprises a nucleotide sequence with no internal complementarityand no complementarity to any other pendant moiety, thereby giving thepervious structure a substantially 0% internal complementarity.

A nucleic acid nanostructure or a component structure thereof (e.g., acompacted structure, a pervious structure) may comprise an internalcomplementarity of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or morethan 99%. Alternatively or additionally, a nucleic acid nanostructure ora component structure thereof may comprise an internal complementarityof no more than about 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than 1%. Insome configurations, a nucleic acid nanostructure or a componentstructure thereof may be considered to have high internalcomplementarity if the internal complementarity exceeds a percentage,such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more than 99%. In some configurations, a nucleic acidnanostructure or a component structure thereof may be considered to havelow internal complementarity if the internal complementarity falls belowa percentage, such as no more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less than 1%.

A nucleic acid nanostructure or a component structure thereof with ahigh internal complementarity may comprise some amount ofsingle-stranded nucleic acid. A nucleic acid nanostructure or acomponent structure thereof with a high internal complementarity maycomprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, or more than 20% single-stranded nucleic acids. In someconfigurations, a nucleic acid nanostructure or a component structurethereof with a high internal complementarity may comprise nosingle-stranded nucleic acids. A nucleic acid nanostructure or acomponent structure thereof with a low internal complementarity maycomprise some amount of double-stranded nucleic acid. A nucleic acidnanostructure or a component structure thereof with a low internalcomplementarity may comprise no more than about 20%, 15%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1% double-stranded nucleicacids. In some configurations, a nucleic acid nanostructure or acomponent structure thereof with a low internal complementarity maycomprise no double-stranded nucleic acids.

A nucleic acid nanostructure may comprise a region comprising lowinternal complementarity (e.g., a pervious structure), in which theregion comprising the low internal complementarity comprises a pluralityof pendant moieties. A pendant moiety may comprise a capture moiety, asset forth herein. A pendant moiety need not comprise a capture moiety. Anucleic acid nanostructure may comprise a plurality of oligonucleotides,in which each oligonucleotide comprises a first nucleotide sequence thathybridizes to a complementary nucleic acid to form a portion of astructure with high internal complementarity, and in which eacholigonucleotide comprises a pendant moiety that does not hybridize tothe region of high internal complementarity. In some cases, a pendantmoiety may comprise a single-stranded oligonucleotide or a non-nucleicacid polymer chain (e.g., polyethylene glycol, polyethylene,polypropylene, etc.). A pendant moiety may comprise a polymer chain(e.g., a nucleic acid chain, a non-nucleic acid polymer chain), in whichthe polymer chain comprises a linear chain, a branched chain, adendrimeric chain, or a combination thereof. In some configurations, apendant moiety of a plurality of pendant moieties may comprise anunbound terminal residue. In some configurations, a pendant moiety of aplurality of pendant moieties may comprise no self-complementarity. Insome configurations, a pendant moiety of a plurality of pendant moietiesmay comprise a homopolymer sequence selected from the group consistingof poly-T, a poly-A, a poly-G, and a poly-C. For example, anoligonucleotide may be extended by an enzyme (e.g., terminaldeoxynucleotidyl transferase) in the presence of a homogeneous pluralityof deoxythymidine nucleotides to form a poly-T sequence on theoligonucleotide. A plurality of pendant moieties may comprise ahomogeneous plurality of pendant moieties, in which each pendant moietycomprises a same chemical structure as each other pendant moiety of theplurality of pendant moieties. A plurality of pendant moieties maycomprise a heterogeneous plurality of pendant moieties, in which a firstpendant moiety comprises a different chemical structure from a secondpendant moiety of the plurality of pendant moieties.

A pendant moiety or a component thereof may comprise a nucleotidesequence (e.g., a homopolymer, a polynucleotide repeat, anoligonucleotide without self-complementarity, an oligonucleotide withself-complementarity, etc.). A nucleotide sequence of a pendant moietyor a component thereof may have a sequence length or chemicalcomposition exemplified herein for staple oligonucleotides.

A nucleic acid nanostructure may comprise a region comprising a lowinternal complementarity (e.g., a pervious structure), in which theregion comprising the low internal complementarity comprises a quantityof pendant moieties. A region comprising a low internal complementaritymay comprise at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 pendantmoieties. Alternatively or additionally, a region comprising a lowinternal complementarity may comprise no more than about 1000, 900, 800,700, 600, 500, 400, 300, 200, 100, 50, 40, 35, 30, 25, 20, 15, 10, 5, orless than 5 pendant moieties. A quantity of pendant moieties of anucleic acid nanostructure may be determined based upon a quantity ofpositions available on a face (e.g., a capture face, a utility face) ofa nucleic acid nanostructure. A quantity of pendant moieties of anucleic acid nanostructure may be determined based upon a desiredsurface density of the pendant moieties on a face of the nucleic acidnanostructure. For example, it may be advantageous to maximize surfacedensity of pendant capture moieties on a capture face of a SNAP, inwhich the pendant capture moieties have a substantially homogeneoussurface density. In such an example, the maximum number of pendantmoieties that can be provided to the SNAP may be limited by a quantityof suitable positions that comprise an orientation in the capture faceand a distance from a nearest suitable position that is within, forexample, about 20%, 15%, 10% 5%, or less than 5% of an average distancebetween suitable positions. A quantity of pendant moieties provided to anucleic acid nanostructure may be determined based upon a strength of adesired interaction with another entity (e.g., an analyte, a nucleicacid nanostructure, a solid support, a reagent, etc.). For example,additional pendant capture moieties may be added to a nucleic acidnanostructure to increase a strength of a coupling interaction with asurface of a solid support.

A nucleic acid nanostructure may comprise a compacted structure. Acompacted structure may comprise a plurality of tertiary structures(e.g., helical double-stranded nucleic acids). Each tertiary structuremay comprise an axis of symmetry (e.g., a helical axis) that defines anangular orientation of the tertiary structure. A distance betweenadjacent or non-adjacent tertiary structures may be measured as adistance between respective axes of symmetry of the tertiary structures.An average distance between adjacent or non-adjacent non-paralleltertiary structures may be measured as an average distance betweenrespective axes of symmetry of the tertiary structures. A compactedstructure may comprise a plurality of tertiary structures, in which aposition, orientation, and/or freedom of motion is constrained between afirst tertiary structure and a second tertiary structure (e.g., anadjacent tertiary structure, a non-adjacent tertiary structure). Aposition, orientation, and/or freedom of motion between a first tertiarystructure and a second tertiary structure may be constrained by one ormore linking strands, as set forth herein.

A compacted structure may comprise a plurality of tertiary structures,in which the plurality of tertiary structures comprises a first tertiarystructure comprising a first axis of symmetry and a second tertiarystructure comprising a second axis of symmetry, in which the firsttertiary structure is adjacent to the second tertiary structure, and inwhich a constrained position of the first tertiary structure relative tothe second tertiary structure comprises an average separation distancebetween the first axis of symmetry and the second axis of symmetry ofless than about 50 nanometers (nm), 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, or less than 2 nm. Alternativelyor additionally, a compacted structure may comprise a plurality oftertiary structures, in which the plurality of tertiary structurescomprises a first tertiary structure comprising a first axis of symmetryand a second tertiary structure comprising a second axis of symmetry, inwhich the first tertiary structure is adjacent to the second tertiarystructure, and in which a constrained position of the first tertiarystructure relative to the second tertiary structure comprises an averageseparation distance between the first axis of symmetry and the secondaxis of symmetry of at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, or more than 50 nm.

A compacted structure may comprise a plurality of tertiary structures,in which the plurality of tertiary structures comprises a first tertiarystructure comprising a first axis of symmetry and a second tertiarystructure comprising a second axis of symmetry, in which the firsttertiary structure is adjacent to the second tertiary structure, and inwhich the constrained position of the first tertiary structure relativeto the second tertiary structure comprises an average angular offset ofat least about 0°, 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°, 100°,110°, 120°, 130°, 140°, 150°, 160°, 170°, or 180° between the first axisof symmetry and the second axis of symmetry. Alternatively oradditionally, a compacted structure may comprise a plurality of tertiarystructures, in which the plurality of tertiary structures comprises afirst tertiary structure comprising a first axis of symmetry and asecond tertiary structure comprising a second axis of symmetry, in whichthe first tertiary structure is adjacent to the second tertiarystructure, and in which the constrained position of the first tertiarystructure relative to the second tertiary structure comprises an averageangular offset of no more than about 180°, 170°, 160°, 150°, 140°, 130°,120°, 110°, 100°, 90°, 80°, 70°, 60°, 50°, 40°, 30°, 20°, 10°, or 0°between the first axis of symmetry and the second axis of symmetry.

A nucleic acid nanostructure may comprise a compacted structure, inwhich the compacted structure comprises a nucleic acid origami, as setforth herein. A nucleic acid origami may comprise one or more faces, inwhich a face of the one or more faces comprises a moiety (e.g., adisplay moiety, a capture moiety, a utility moiety, etc.), and in whichthe nucleic acid origami provides a tunable location and/or orientationfor the moiety. In some configurations, a nucleic acid origami maycomprise a first face and a second face, in which the first face isoffset from the second face by an average angle of at least about 30°,45°, 60°, 90°, 120°, 135°, 150°, 160°, 170°, or 180°. Alternatively oradditionally, a nucleic acid origami may comprise a first face and asecond face, in which the first face is offset from the second face byan average angle of no more than about 180°, 170°, 160°, 150°, 135°,120°, 90°, 60°, 45°, 30°, or less than 30°. A nucleic acid origami maycomprise a first face and a second face, in which the first facecomprises a display moiety, as set forth herein, and the second face isadjacent to a pervious structure. A nucleic acid origami may comprise afirst face and a second face, in which the first face comprises adisplay moiety, as set forth herein, and the second face is coupled to apervious structure. For example, a nucleic acid origami with a tilestructure may comprise a first face that comprises a click-type reactiongroup that is configured to couple an analyte, and a second face thatcomprises a capture moiety comprising a plurality of pendant moieties,in which a pervious structure comprises the plurality of pendantmoieties, and in which the first face is substantially opposite inorientation from the second face (e.g., about 180° offset).

A nucleic acid nanostructure may comprise a compacted structure and apervious structure, in which the pervious structure comprises a spatialdistribution with respect to the compacted structure. A spatialdistribution may comprise an isotropic distribution or an anisotropicdistribution. A spatial distribution may be with respect to two spatialdimensions and/or three spatial dimensions. For example, a perviousstructure may comprise an isotropic spatial distribution in two spatialdimensions but an anisotropic spatial distribution with respect to threespatial distributions. For example, FIG. 52A depicts a cross-sectionalview of a nucleic acid nanostructure with a compacted structure 5210that is coupled to a pervious structure comprising a plurality ofpendant moieties 5212. With respect to a plane of symmetry 5250 centeredat an average midline of the compacted structure 5210, the plurality ofpendant moieties 5212 are confined to a volume 5230 that is entirelybelow the plane of symmetry 5250 (e.g., anisotropic with respect to theplane of symmetry 5250). FIG. 52B depicts a top-down view of the nucleicacid nanostructure depicted in FIG. 52A. From the top-down view, theplurality of pendant moieties have a substantially isotropic spatialdistribution with respect to a center point of the compacted structure5210. In some configurations, a spatial distribution of a perviousstructure relative to a compacted structure may be determined withrespect to an imaginary volume (e.g., a sphere, a hemisphere, a cube, acylinder, etc.) that fully encloses a nucleic acid nanostructurecontaining the compacted structure and the pervious structure. Inparticular configurations, an imaginary volume may be positioned withrespect to an alignment of a compacted structure, such as an axis ofsymmetry, a plane of symmetry, or a face of the compacted structure. Insome configurations, an anisotropic volumetric distribution may comprisea fraction of a hemispherical volume surrounding the compacted structurethat does not comprise the pervious structure. In some configurations,an anisotropic volumetric distribution may comprise a fraction of aspherical volume surrounding the compacted structure excluding a volumecomprising an analyte of interest coupled to the compacted structure.

A nucleic acid nanostructure may comprise a compacted structure and apervious structure, in which the compacted structure and the perviousstructure each occupy a characteristic volume, in which thecharacteristic volume comprises a minimum, average, or maximum volumeoccupied by structure on a spatial and/or temporal basis. A volume of acompacted structure and/or a pervious structure may vary depending uponthe configuration of a nucleic acid nanostructure comprising thecompacted structure and/or pervious structure (e.g., bound to a solidsupport, unbound to a solid support, coupled to an analyte, coupled to areagent, etc.). For example, a nucleic acid nanostructure comprising acompacted structure and a pervious structure may bind to a solid supportby a capture moiety comprising a pervious structure, in which the volumeof the compacted structure is unchanged by the binding but the volume ofpervious structure decreases due to the binding. In some configurations,an average volume of a compacted structure need not vary according to aconfiguration of a nucleic acid nanostructure comprising the compactedstructure. In some configurations, a volume occupied by a compactedstructure may be larger than a volume occupied by a pervious structure.In other configurations, a volume occupied by a pervious structure maybe larger than a volume occupied by a compacted structure.

A nucleic acid nanostructure may comprise an average effective surfacearea (e.g., a nucleic acid nanostructure in solution) and/or footprint(e.g., a nucleic acid nanostructure coupled to a solid support). Anucleic acid nanostructure may comprise a compacted structure and/or apervious structure, in which the compacted and/or the pervious structurecomprises an average effective surface area and/or footprint. An averageeffective surface area and/or footprint of a compacted structure and/ora pervious structure may be modified, for example, to modulate thestrength of an interaction with another entity (e.g., an analyte, anucleic acid nanostructure, a solid support, a reagent, etc.). In someconfigurations, an effective surface area and/or footprint of a perviousstructure may be substantially the same as an effective surface areaand/or footprint of a nucleic acid nanostructure. In otherconfigurations, an effective surface area of a pervious structure may besmaller than an effective surface area of a nucleic acid nanostructure.In some configurations, an effective surface area of a perviousstructure may be smaller than an effective surface area of a compactedstructure. In some configurations, an effective surface area of apervious structure may be larger than an effective surface area of acompacted structure. In some configurations, a footprint of a nucleicacid nanostructure may be larger than an effective surface area of anucleic acid nanostructure. In other configurations, a footprint of anucleic acid nanostructure may be less than or equal to an effectivesurface area of a nucleic acid nanostructure. In some configurations, afootprint of a compacted structure may be less than or equal to aneffective surface area of a compacted structure. In some configurations,a nucleic acid nanostructure may comprise a footprint, in which thefootprint of the nucleic acid nanostructure is the greater than, equalto, or less than an effective surface area of the nucleic acidnanostructure.

Nucleic Acids at Solid Supports

A nucleic acid, as set forth herein, may be configured to couple with asolid support or a site thereof, as set forth herein. In someconfigurations, a plurality of nucleic acids may be coupled to a solidsupport, in which each nucleic acid is configured to couple an analyteof interest to the solid support, thereby forming an array of analytesof interest on the solid support. A nucleic acid may be configured intandem with a solid support or a surface thereof to increase alikelihood of one or more outcomes of a nucleic acid/solid supportinteraction, including: 1) binding a nucleic acid to an address of thesolid support that is configured to bind the nucleic acid, 2) inhibitinga binding of a nucleic acid to an address of the solid support that isnot configured to bind the nucleic acid, 3) inhibiting binding of asecond nucleic acid to an address comprising a first nucleic acid, inwhich the address is not configured to bind a second nucleic acid; 4)inhibiting an improper binding orientation of a nucleic acid, and 5)displaying an analyte of interest in an accessible fashion for anarray-based process (e.g., a characterization assay, a synthesisprocess, etc.).

Systems of nucleic acids and solid supports may be configured to producearrays of analytes with a substantially uniform surface density ofanalytes of interest. Of particular interest are systems of nucleicacids and solid supports that produce high-density arrays of analytes ofinterest, for example, in which each analyte of the array of analytes isindividually resolvable at single-analyte resolution. An array ofanalytes of interest may comprise one or more properties of: i)comprising a maximal number, density or pitch of individually resolvablearray addresses containing one and only one analyte of interest, ii)comprising a minimal number, density or pitch of individually resolvablearray addresses containing two or more analytes of interest, iii)comprising a minimal number, density or pitch of individually resolvablearray addresses containing no analytes of interest, and iv) comprising amaximal number, density or pitch of individually resolvable arrayaddresses containing no analytes of interest. A useful array of analytesof interest for a single-analyte process may comprise a spatialdistribution (e.g. pitch or density) of single analytes at arrayaddresses, in which the spatial distribution contains a higher amount ofsites occupied by one and only one single analyte with reference to astatistical distribution, such as a Poisson distribution or a normaldistribution. For example, given a system of a solid support containingN analyte binding sites and a plurality of N nucleic acids coupled toanalytes contacted with the solid support, or a method for making such asystem, in which neither the solid support nor the nucleic acids bias alikelihood of a nucleic acid binding to any particular analyte bindingsite, a Poisson distribution would predict ˜37% of the N analyte bindingsites containing no deposited analytes of interest, ˜37% of the Nanalyte binding sites containing one and only one deposited analyte ofinterest, and ˜26% of the N analyte binding sites containing two or moredeposited analytes of interest. Accordingly, it is advantageous toconfigure systems of nucleic acids and solid supports or methods formaking such systems that provide single-analyte occupancy at least 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than 99% of sites ofan array. Alternatively or additionally, it is advantageous to configuresystems of nucleic acids and solid supports that provide: 1) a maximizedratio of sites with single analyte occupancy:sites with no analyteoccupancy, and/or 2) a maximized ratio of sites with single analyteoccupancy:sites with multiple analyte occupancy.

The skilled person will readily recognize innumerable combinations ofsolid supports, as set forth herein, and nucleic acids as set forthherein, for forming arrays of single analytes. In some configurations,an array-forming system may comprise a nucleic acid comprising one ormore capture moieties and a solid support comprising one or moresurface-linked moieties, in which the nucleic acid is configured to bebound to the solid support by a coupling interaction of the one or morecapture moieties with the one or more surface-linked moieties. A capturemoiety may be selected for one or more properties of: 1) forming aspecific interaction with a surface-linked moiety of a solid support,optionally in a rapid fashion (high kinetic on-rate), 2) forming aspecific interaction with a long-duration to a surface-linked moiety ofa solid support (low kinetic off-rate), 3) not forming low specificitybinding interactions with other entities in the system (e.g., othernucleic acids, analytes coupled to other nucleic acids, non-bindingregions of the array, etc.), and 4) providing a physical and/or chemicalproperty that inhibits binding of other nucleic acids at an array site(e.g., steric occlusion, electrostatic repulsion, magnetic repulsion,etc.). A surface-linked moiety may be selected for one or moreproperties of: 1) forming a specific interaction with a capture moietyof a nucleic acid, optionally in a rapid fashion (high kinetic on-rate),2) forming a specific interaction with a long-duration to a capturemoiety of a nucleic acid (low kinetic off-rate), 3) inhibiting bindinginteractions with other entities in the system (e.g., analytes), 4)providing a physical and/or chemical property that inhibits binding ofother nucleic acids at an array site (e.g., steric occlusion,electrostatic repulsion, magnetic repulsion, etc.), and 5) facilitatingbinding of a nucleic acid in a specific location and orientation (e.g.,centered symmetrically on a site with an analyte of interest not incontact with a solid support).

Surprisingly, a system of nucleic acids and a solid support, or a methodfor making such a system, may be configured to obtain spatial control ofnucleic acid binding locations on single-analyte arrays through theformation of weak binding interactions between one or more capturemoieties of a nucleic acid and one or more surface-linked moieties ofthe solid support. Commonly, molecules are coupled to surfaces throughthe formation of strong binding interactions (e.g., click-type covalentbonds, streptavidin-biotin coupling, etc.). Such strong bindinginteractions are advantageous for permanently coupling a molecule to asurface; however, if the molecule initially binds toward an edge of abinding site, sufficient additional space may exist at the binding siteto couple one or more additional molecules. In contrast, a system ofnucleic acids and a solid support may be configured to obtain spatialcontrol of nucleic acid binding locations on single-analyte arraysthrough a multi-valency affect in which a plurality of weak bindinginteractions between one or more capture moiety and a plurality ofsurface-linked moieties provide a binding strength comparable to asingle strong binding interaction while permitting a nucleic acid tospatially re-arrange on a solid support from an initial bindingconfiguration to a more stable final binding configuration. Withoutwishing to be bound by theory, a stable binding configuration of anucleic acid comprising one or more capture moieties may be obtained ona binding site comprising an excess of surface-linked moieties dueto: 1) energetic favorability of specific binding interactions betweenthe one or more capture moieties and the excess of surface-linkedmoieties, and 2) entropic favorability caused by numerous possibleconfigurations of binding between the one or more capture moieties andthe excess of surface-linked moieties.

FIGS. 58A-58C illustrate a concept of achieving a stable configurationof a nucleic acid nanostructure on a surface through a multi-valentbinding interaction. FIG. 58A illustrates a solid support 5800comprising a site 5801 with a plurality of surface-linked moieties 5805that are configured to couple to complementary capture moieties 5835 ofa nucleic acid nanostructure 5830. The nucleic acid nanostructure 5830is optionally coupled to an analyte 5840. FIG. 58B shows an initialconfiguration of the nucleic acid nanostructure 5830 upon contact of thenucleic acid nanostructure 5830 with the solid support 5800 at a randomlocation of the site 5801. Due to the location of contact, only onecoupling interaction has occurred between a surface-linked moiety 5805and a capture moiety 5835. FIG. 58C depicts a more stable finalconfiguration of the nucleic acid nanostructure 5830 after a spatialrearrangement on the surface of the site 5801. The final configurationmay be more stable than the initial configuration due to the increasedquantity of coupling interactions between surface-linked moieties 5805and capture moieties 5835. The final configuration may also be morestable than the initial configuration because it has other possiblecombinations of couplings between surface-linked moieties 5805 andcapture moieties 5835 that the structure can re-arrange into if anycouping between a surface-linked moiety 5805 and a capture moiety 5835is disrupted.

FIGS. 52A-52H and 53A-53D illustrate configurations of nucleic acidnanostructures that may form multi-valent binding interactions with asolid support or a surface thereof. The nucleic acid nanostructuresdepicted in FIGS. 53A-53D comprise a plurality of pendantoligonucleotides that are configured to form hybridization bindinginteractions with complementary oligonucleotides of a solid support.Additional configurations of nucleic acid nanostructures that formmulti-valent binding interactions are depicted in FIGS. 55A-55D. FIG.55A depicts a nucleic acid nanostructure 5530 that is coupled to ananalyte 5540. The nucleic acid nanostructure comprises a first internalsingle-stranded nucleic acid 5532 and a second internal single-strandednucleic acid 5534 that are configured to couple to a first complementarysurface-linked oligonucleotide 5520 and a second surface-linkedoligonucleotide 5522, each of which is coupled to a site 5501 of a solidsupport 5500 in a molar excess relative to available binding sites ofthe nucleic acid nanostructure 5530. FIG. 55B depicts the nucleic acidnanostructure 5530 in a coupled configuration at the site 5501 of thesolid support 5500. The first complementary surface-linkedoligonucleotide 5520 and the second surface-linked oligonucleotide 5522have coupled to the first internal single-stranded nucleic acid 5532 andthe second internal single-stranded nucleic acid 5534. Excesssurface-linked oligonucleotides 5520 and 5522 remain, therebyfacilitating spatial re-arrangement of the nucleic acid nanostructure5530 by re-arrangement of binding interactions if favorable. FIG. 55Cdepicts a nucleic acid nanostructure 5530 that is coupled to an analyte5540. The nucleic acid nanostructure comprises a plurality of capturemoieties 5550 (e.g., antibodies, antibody fragments, aptamers, etc.)that are configured to couple to a plurality of surface-linked bindingligands 5555, each of which is coupled to a site 5501 of a solid support5500 in a molar excess relative to available capture moieties of thenucleic acid nanostructure 5530. The site 5501 may further comprise aplurality of non-coupling moieties 5560 (e.g., passivating moieties thatprevent non-specific binding). FIG. 55D depicts the nucleic acidnanostructure 5530 in a coupled configuration at the site 5501 of thesolid support 5500. The plurality of capture moieties 5550 have coupledto the plurality of surface-linked binding ligands 5555. Excesssurface-linked binding ligands 5555 remain, thereby facilitating spatialre-arrangement of the nucleic acid nanostructure 5530 by re-arrangementof binding interactions if favorable. The configurations depicted inFIGS. 52A-52H and 53A-53D contain various chemical structures andspatial configurations of capture moieties comprising a perviousstructure (e.g., a plurality of pendant moieties). An advantageousnucleic acid nanostructure may comprise a pervious structure comprisinga plurality of capture moieties, in which each capture moiety isconfigured to form a binding interaction with a surface-linked moiety ofa solid support. An advantageous nucleic acid nanostructure may comprisea pervious structure comprising a capture moiety, in which the capturemoiety is configured to form a plurality of binding interactions with aplurality of surface-linked moieties of a solid support.

FIGS. 56A-56C depict examples of differing systems of nucleic acidnanostructures coupled to solid supports. FIG. 56A shows a solid support5600 comprising a site 5601, in which the site comprises a plurality ofsurface-linked oligonucleotides 5605 comprising poly-A sequences. Anucleic acid nanostructure 5630 is coupled to an analyte 5640, and isfurther coupled to the site 5601 by a pervious structure comprising aplurality of pendant oligonucleotides 5635 comprising poly-T sequences.Each pendant oligonucleotide 5635 is sufficiently long to couple tomultiple surface-linked oligonucleotides 5605, thereby forming amulti-valent binding interaction between the site 5601 and the nucleicacid nanostructure 5630. Optionally, the plurality of pendantoligonucleotides 5635 may comprise oligonucleotides of differing chainlengths. FIG. 56B depicts a similar composition to FIG. 56A, however thenucleic acid nanostructure 5630 instead comprises a pervious structurecomprising a plurality of oligonucleotide loops 5637 comprising poly-Tsequences. Each pendant oligonucleotide loop 5637 is sufficiently longto couple to multiple surface-linked oligonucleotides 5605, therebyforming a multi-valent binding interaction between the site 5601 and thenucleic acid nanostructure 5630. FIG. 56C shows a solid support 5600comprising a site 5601, in which the site comprises a first plurality ofsurface-linked oligonucleotides 5605 comprising poly-A sequences and asecond plurality of surface-linked oligonucleotides 5606 comprisingcomplementarity to a heteropolymeric nucleotide sequence 5636. A nucleicacid nanostructure 5630 is coupled to an analyte 5640, and is furthercoupled to the site 5601 by a pervious structure comprising a pluralityof pendant oligonucleotides 5635 comprising poly-T sequences and furthercontaining intermediate nucleotide sequences comprising thenon-repeating nucleotide sequence 5636. The number of heteropolymericnucleotide sequences 5636 or complementary surface-linkedoligonucleotides 5606 may be limited to reduce the number of re-arrangedconfigurations available to a nucleic acid nanostructure coupled to asolid support.

A coupling of a nucleic acid nanostructure to a solid support or asurface thereof may cause a conformational change of the nucleic acidnanostructure. In some configurations, a nucleic acid nanostructure maycomprise a compacted structure and a pervious structure, in whichcoupling of the nucleic acid nanostructure to a solid support or asurface thereof causes no substantial change in conformation (e.g.,shape, volume, effective surface area, footprint, etc.) to the compactedstructure, and in which coupling of the nucleic acid nanostructure to asolid support or a surface thereof causes a substantial change inconformation (e.g., shape, volume, effective surface area, footprint,etc.) to the pervious structure. FIG. 57 depicts a change inconformation associated with a binding of a nucleic acid nanostructurecomprising a compacted structure 5710 and a pervious structure 5720 whenthe nanostructure binds to a site 5701 of a solid support 5700. In aninitial unbound configuration, the compacted structure 5710 comprises awidth L_(C,i), a thickness H_(C,i), and a volume V_(C,i) and thepervious structure 5720 comprises a width L_(N,i), a thickness H_(N,i),and a volume V_(N,i). After coupling to the surface, the perviousstructure may become compressed and elongated due to the pendantmoieties forming a maximal number of binding interactions with a surfaceof the site 5701. Accordingly, in the final bound configuration, thecompacted structure 5710 may comprise a width L_(C,f), a thicknessH_(C,f), and a volume V_(C,f), in which the values are substantiallyunchanged from the initial values. In contrast, the pervious structure5720 may comprise a width L_(N,f), a thickness H_(N,f), and a volumeV_(N,f), in which a final value for the width has increased relative tothe initial value, a final value for the height has decreased relativeto the initial value, and a final value for the volume may or may notchange depending upon the nature of the binding interactions with thesite 5701. Nucleic acid nanostructures that have conformational changesmay be advantageous for increasing a footprint of the nucleic acidnanostructure on the surface area of the binding site, therebydecreasing available surface area for binding of other nucleic acidnanostructures or other entities.

In an aspect, provided herein is a composition, comprising: a) a solidsupport comprising a plurality of sites, and b) a plurality of nucleicacid nanostructures (e.g., SNAPs), in which each nucleic acidnanostructure is coupled to, or configured to couple to, an analyte, andin which each nucleic acid nanostructure of the plurality of nucleicacid nanostructures is coupled to a site of the plurality of sites, inwhich the plurality of sites comprises a first subset comprising a firstquantity of sites and a second subset comprising a second quantity ofsites, in which each site of the first subset comprises two or morecoupled nucleic acid nanostructures, in which each site of the secondsubset comprises one and only one coupled nucleic acid nanostructure,and in which a ratio of the quantity of sites of the first subset to thequantity of sites of the second subset is less than a ratio predicted bya Poisson distribution.

In another aspect, provided herein is an analyte array, comprising: a) asolid support comprising a plurality of sites; and b) a plurality ofnucleic acid nanostructures (e.g., SNAPs), in which each nucleic acidnanostructure is coupled to an analyte of interest, and in which eachnucleic acid nanostructure of the plurality of nucleic acidnanostructures is coupled to a site of the plurality of sites, in whichat least 40% of sites of the plurality of sites comprise one and onlyone analyte of interest.

In another aspect, provided herein is a composition, comprising: a) asolid support comprising a site that is configured to couple a nucleicacid nanostructure, and b) the nucleic acid nanostructure, in which thenucleic acid nanostructure is coupled to the site, in which the nucleicacid nanostructure is coupled to an analyte of interest; and in whichthe nucleic acid nanostructure is configured to prevent contact betweenthe analyte of interest and the solid support.

In another aspect, provided herein is a composition, comprising: a) asolid support comprising a site that is configured to couple a nucleicacid nanostructure, wherein the site comprises a surface area; and b)the nucleic acid nanostructure, in which the nucleic acid nanostructureis coupled to the site, in which the nucleic acid nanostructure iscoupled to, or configured to couple to, an analyte of interest; in whichthe nucleic acid nanostructure comprises a total effective surface areain an unbound configuration, in which the nucleic acid nanostructurecomprises a compact structure with an effective surface area in theunbound configuration, in which the effective surface area of thecompacted structure is less than 50% of the surface area of the site,and in which the unbound configuration comprises the nucleic acidnanostructure being uncoupled to the site.

An array may comprise a plurality of sites, in which a site has adeterminable occupancy. When used in reference to a site of an array,occupancy may refer to a detected or inferred presence of an entity(e.g., a nucleic acid, an analyte, a nucleic acid and an analyte, anucleic acid coupled to an analyte, a nucleic acid or an analyte, etc.)at the array site. In particular instances, occupancy may further referto a property (e.g., a chemical property, a physical property, etc.) orcharacteristic (e.g., a spatial orientation, a temporal orientation, abound state, an unbound state, etc.) of an entity that is detected orinferred to be present at an array site. For example, when forming anarray for a polypeptide assay, a complex comprising a polypeptidecoupled to a nucleic acid nanostructure may deposit on an array site bya coupling of the polypeptide to the array site rather than the nucleicacid to the array site, thereby rendering the polypeptidenon-interrogable during the polypeptide assay. In such a case, the sitemay be considered unoccupied by an analyte due to the orientation of thecomplex on the array site. When used in reference to an array comprisinga plurality of sites, an occupancy may refer to a percentage or fractionof sites of the plurality of sites comprising a detected or inferredpresence of an entity (e.g., e.g., a nucleic acid, an analyte, a nucleicacid and an analyte, a nucleic acid coupled to an analyte, a nucleicacid or an analyte, etc.). In particular instances, occupancy mayfurther refer to a percentage or fraction of sites of the plurality ofsites containing a detected or inferred presence of an entity with aproperty (e.g., a chemical property, a physical property, etc.) orcharacteristic (e.g., a spatial orientation, a temporal orientation, abound state, an unbound state, etc.). For example, an array may have adetectable analyte occupancy fraction of 0.9 if 9 sites of every 10sites contain a detectable analyte. In some configurations, occupancymay refer to a quantity of entities at a site, such as about 0, 1, 2, 3,4, 5, or more entities at a site. In some configurations, occupancy mayrefer to a quantity of entities with a particular property orcharacteristics at a site, such as about 0, 1, 2, 3, 4, 5, or moreentities at a site.

Accordingly, an array of analytes may be characterized by a quantitativecomparison of two or more measures of occupancy. For example, it may beuseful to compare an occupancy of sites of a plurality of sites of anarray containing no analytes to an occupancy of sites of the pluralityof sites of the array containing at least one analyte. In anotherexample, it may be useful to compare an occupancy of sites of aplurality of sites of an array containing one and only one analyte to anoccupancy of sites of the plurality of sites of the array containing twoor more analytes. In some configurations, a comparison of two or moremeasures of occupancy may provide a useful quality controlcharacteristic after forming an array of analytes. For example, an arrayof analytes may be rejected for further use if a ratio of sites with anoccupancy of two or more analytes to sites with an occupancy of one andonly one site exceeds a threshold value, such as a ratio predicted by aPoisson distribution. Table I lists pairs of measures of occupancy whoseratios may be useful for characterizing an array, as set forth herein.

TABLE I 1^(st) Occupancy 2^(nd) Occupancy Critical Ratio Measure Measureof 1^(st) to 2^(nd) Sites occupied Sites unoccupied >1.72 Sites w/only 1analyte Sites w/2+ analytes >1.40 Sites w/only 1 nucleic Sites w/2+nucleic acid >1.40 acid nanostructure nanostructures Sites w/1+ analyteSites w/0 analytes >1.72 Sites w/1+ nucleic acid Sites w/0 nucleicacid >1.72 nanostructure nanostructures Sites w/1+ detectable Sites w/0detectable >1.72 analyte analytes

In another aspect, provided herein is a method of characterizing anarray of analytes, comprising: a) providing an array of analytes, as setforth herein, b) determining a first measure of occupancy for the arrayof analytes, as set forth herein, c) determining a second measure ofoccupancy for the array of analytes, as set forth herein, and d)comparing a ratio of the first measure of occupancy to the secondmeasure of occupancy to an array criterium. In some configurations, anarray criterium may comprise a ratio of a first measure of occupancy toa second measure of occupancy for a hypothetical array of analytes withan occupancy distribution that fits a statistical or stochasticdistribution (e.g., a Poisson distribution, a normal distribution, abinomial distribution, etc.). For example, an array criterium maycomprise a critical ratio listed in Table I, or any other conceivableratio of measures of occupancy. In some configurations, a ratio of afirst measure of occupancy to a second measure of occupancy may meet orexceed an array criterium predicted by a statistical or stochasticdistribution (e.g., a Poisson distribution). In other configurations, aratio of a first measure of occupancy to a second measure of occupancymay not meet or exceed an array criterium predicted by a statistical orstochastic distribution (e.g., a Poisson distribution). A method ofcharacterizing an array of analytes may further comprise a step of,based upon comparing a ratio of a first measure of occupancy to a secondmeasure of occupancy to an array criterium, discarding the array ofanalytes. For example, an array of analytes with a level of analyteoccupancy beneath an array criterium may be discarded. A method ofcharacterizing an array of analytes may further comprise a step of,based upon comparing a ratio of a first measure of occupancy to a secondmeasure of occupancy to an array criterium, separating the analytes fromthe array of analytes. For example, an array of analytes with an analyteoccupancy beneath an array criterium may be contacted with a strippingmedium (e.g., a denaturant, a chaotrope) to remove coupled analytesand/or nucleic acids before reforming the array of analytes with a newplurality of analytes. A method of characterizing an array of analytesmay further comprise a step of, based upon comparing a ratio of a firstmeasure of occupancy to a second measure of occupancy to an arraycriterium, providing additional analytes to the array of analytes. Forexample, an array of analytes with a level of analyte occupancy beneathan array criterium may be contacted with additional analytes coupled tonucleic acids to increase the analyte occupancy. A method ofcharacterizing an array of analytes may further comprise a step of,based upon comparing a ratio of a first measure of occupancy to a secondmeasure of occupancy to an array criterium, utilizing the array ofanalytes in an array-based process (e.g., an assay, a synthesis, etc.).

In some configurations, an array may comprise a plurality of sites, inwhich the plurality of sites comprise a first subset of sites, in whicheach site of the first subset comprises a first measure of occupancy(e.g., quantity of entities coupled to the array site, presence of anentity, presence of a detectable entity, etc.), a second subset ofsites, in which each site of the second subset comprises a secondmeasure of occupancy, and optionally a third subset of sites, in whicheach site of the third subset comprises a third measure of occupancy.Occupancy of an array may be determined by a method such as fluorescencemicroscopy, electron microscopy, atomic force microscopy, etc. An arraymay comprise a plurality of sites, in which at least about 10%, 20%,30%, 35%, 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, or more than99.99999% of the sites of the plurality of sites comprise an occupancyof at least one analyte. Alternatively or additionally, an array maycomprise a plurality of sites, in which no more than about 99.99999%,99.9999%, 99.999%, 99.99%, 99.9%, 99%, 95%, 90%, 85%, 80%, 75%, 70%,65%, 60%, 55%, 50%, 45%, 40%, 37%, 35%, 30%, 20%, 10%, or less than 10%of the sites of the plurality of sites comprise an occupancy of at leastone analyte. An array may comprise a plurality of sites, in which atleast about 10%, 20%, 30%, 35%, 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%,99.99999%, or more than 99.99999% of the sites of the plurality of sitescomprise an occupancy of no more than one analyte. Alternatively oradditionally, an array may comprise a plurality of sites, in which nomore than about 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99%, 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 37%, 35%, 30%,20%, 10%, or less than 10% of the sites of the plurality of sitescomprise an occupancy of no more than one analyte.

Continuing with the example of an array comprising a first subset ofsites comprising an occupancy of two or more analytes, a second subsetof sites comprising an occupancy of one analyte, and a third subset ofsites comprising an occupancy of zero analytes, a ratio of a quantity ofsites of the first subset to a quantity of sites of the second subset,or a ratio of a quantity of sites of the third subset to a quantity ofsites of the second subset may substantially conform to a ratiopredicted by a probabilistic or stochastic distribution, such as aPoisson distribution, normal distribution, binomial distribution, etc. Aratio of a quantity of sites of the first subset to a quantity of sitesof the second subset, or a ratio of a quantity of sites of the thirdsubset to a quantity of sites of the second subset may deviate from aratio predicted by a probabilistic or stochastic distribution. A ratioof quantity of sites of the first subset to quantity of sites of thesecond subset may have a value of no more than about 0.71, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0001, 0.00001,0.000001, or less than 0.000001. Alternatively or additionally, a ratioof quantity of sites of the first subset to quantity of sites of thesecond subset may have a value of at least about 0.000001, 0.00001,0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.71, or more than 0.71. A ratio of quantity of sites of the thirdsubset to quantity of sites of the second subset may have a value of nomore than about 0.99, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05,0.01, 0.005, 0.001, 0.0001, 0.00001, 0.000001, or less than 0.000001.Alternatively or additionally, a ratio of quantity of sites of the thirdsubset to quantity of sites of the second subset may have a value of atleast about 0.000001, 0.00001, 0.0001, 0.001, 0.005, 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.99, or more than 0.99.

Nucleic acid nanostructures or nucleic acid nanostructure complexes maybe characterized by a spacing or separation between analyte couplingsites on adjacent nucleic acid nanostructures or nucleic acidnanostructure complexes in an array of nucleic acid nanostructures ornucleic acid nanostructure complexes. Nucleic acid nanostructures ornucleic acid nanostructure complexes may have a nearest neighborseparation of at least about 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, or more than 10 μm, relative toan adjacent nucleic acid nanostructure or nucleic acid nanostructurecomplex. Alternatively or additionally, nucleic acid nanostructures ornucleic acid nanostructure complexes may have a nearest neighborseparation of no more than about 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm,900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm,250 nm, 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 95 nm, 90 nm, 85nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5 nm, or less than 5 nm, relativeto an adjacent nucleic acid nanostructure or nucleic acid nanostructurecomplex. Nucleic acid nanostructure or nucleic acid nanostructurecomplex nearest neighbor separation may be determined by an opticalmethod such as fluorescence microscopy. In some cases, a nucleic acidnanostructure or nucleic acid nanostructure complex nearest neighborseparation may be calculated as an average value based on, for example,a total fluorescence count over a fixed image area, where the totalfluorescence count may be correlated to a number of observed nucleicacid nanostructures or nucleic acid nanostructure complexes. In othercases, an optical detection system may have sufficient opticalresolution and sensor pixel density to distinguish individual nucleicacid nanostructures or nucleic acid nanostructure complexes anddetermine separation from all nearest neighbor nucleic acidnanostructures or nucleic acid nanostructure complexes.

A nucleic acid nanostructure, as set forth herein, may be configured tocouple with a site of an array and thereby occlude coupling of a secondnucleic acid nanostructure to the site. In some configurations,occluding binding may comprise inhibiting transport of a second nucleicacid nanostructure to the site of the array by a first nucleic acidnanostructure. For example, occluding binding may comprise inhibitingdeposition of a second nucleic acid nanostructure at an array siteduring deposition of a first nucleic acid nanostructure at the arraysite. In some configurations, occluding binding may comprise inhibitingdeposition of a second nucleic acid nanostructure at an array site aftera first nucleic acid nanostructure has coupled to the array site.

A first nucleic acid nanostructure may occlude binding of a secondnucleic acid nanostructure at an array site by occupying a significantportion of a surface area of the array site. In some configurations, anucleic acid nanostructure complex (e.g., a SNAP complex) may be coupledto an array site, in which the nucleic acid nanostructure complexcomprises a coupled plurality of nucleic acid nanostructures, and inwhich, optionally, the nanostructure complex is coupled to, orconfigured to couple to, a single analyte of interest. In otherconfigurations, a nucleic acid nanostructure comprising a perviousstructure may be coupled to an array site, in which the perviousstructure is configured to occlude binding of a second nucleic acidnanostructure to the array site, and in which, optionally, thenanostructure is coupled to, or configured to couple to, a singleanalyte of interest. In some configurations, a pervious structure maycomprise an oligonucleotide that is configured to occlude binding of asecond nucleic acid nanostructure to an array site. Exemplarycompositions for the pervious structure are set forth elsewhere herein,for example, in the context of capture moieties, pendant moieties andpendant oligonucleotides.

A nucleic acid nanostructure coupled to a solid support may beconfigured to inhibit or prevent contact between an analyte of interestand a solid support. In some configurations, a nucleic acidnanostructure may be configured to inhibit or prevent contact between ananalyte of interest and a solid support during deposition of the nucleicacid nanostructure at an array site on the solid support. For example, anucleic acid nanostructure may prevent coupling of the analyte directlyto the surface by a non-specific binding interaction. In otherconfigurations, a nucleic acid nanostructure may be configured toinhibit or prevent contact between an analyte of interest and a solidsupport after deposition of the nucleic acid nanostructure at an arraysite on the solid support. For example, a nucleic acid nanostructure maycomprise a linking moiety that couples an analyte to the nucleic acidnanostructure, in which the linking moiety facilitates an increasedspatial range of motion for the analyte, and in which the nucleic acidnanostructure further comprises a footprint on an array site thatoccludes any surface area of the array site that the analyte couldaccess due to its increased range of motion. In some configurations, anucleic acid nanostructure may comprise a pervious structure, in whichthe pervious structure comprises a moiety that is configured to preventcontact between an analyte of interest and a solid support. In someconfigurations, a pervious structure comprises a moiety that isconfigured to prevent contact between an analyte of interest and a solidsupport by steric occlusion of the solid support. In particularconfigurations, a moiety that is configured to prevent contact betweenan analyte of interest and a solid support comprises a chemical and/orphysical property that is configured to prevent contact between theanalyte of interest and the solid support. In particular configurations,a moiety that is configured to prevent contact between an analyte ofinterest and a solid support comprises an electrically-repulsive moiety,a magnetically-repulsive moiety, a hydrophobic moiety, a hydrophilicmoiety, an amphipathic moiety, or a combination thereof.

An array site on a solid support may be configured to prevent binding ofan analyte to the array site or prevent deposition of more than onenucleic acid nanostructure at the array site. An array site may comprisea moiety that is configured to prevent coupling of an analyte ofinterest to the site or prevent deposition of more than one nucleic acidnanostructure at the array site. In some configurations, a moiety thatis configured to prevent coupling of an analyte of interest to the siteor prevent deposition of more than one nucleic acid nanostructure at thearray site may comprise (i) an oligonucleotide, (ii) a polymer chain,selected from the group consisting of a linear polymer chain, a branchedpolymer chain, and a dendrimeric polymer chain, (iii) a moiety thatcomprises a chemical property that is configured to prevent contactbetween the analyte of interest and the solid support, or (iv) a moietythat comprises an electrically-repulsive moiety, amagnetically-repulsive moiety, a hydrophobic moiety, a hydrophilicmoiety, an amphipathic moiety, or a combination thereof. In someconfigurations, an array site may comprise a first moiety and a secondmoiety, in which the first moiety and the second moiety are configuredto prevent coupling of an analyte of interest to the site or preventdeposition of more than one nucleic acid nanostructure at the arraysite, and in which the first moiety and the second moiety comprise adissimilar chemical structure or a dissimilar property. For example, anarray site may comprise a plurality of polymer chains, in which theplurality of chains comprise a mixture of polymer chains with differingstructures, such as linear polymer chains (e.g., linear PEG, lineardextrans) and branched polymer chains (e.g., branched PEG, brancheddextrans). In another example, an array site may comprise a mixture ofpolymer chains with differing physical properties, such as a mixture ofpolar chains (e.g., PEG chains) and non-polar chains (e.g., polyethylenechains).

A nucleic acid nanostructure may comprise a compacted structure (e.g., anucleic acid origami) that comprises a smaller effective surface areathan a surface area of an array site to which the nucleic acidnanostructure is configured to be coupled. For example, a square,tile-shaped DNA origami may have side lengths of approximately 83nanometers, such that the DNA origami would occupy less than 10% of thesurface area of a 300 nanometer-wide, circular array site if the origamiwas coupled to the array site on one of its square faces. A nucleic acidnanostructure comprising a compacted structure may be configured tooccupy a larger surface area of an array site than an effective surfacearea of the compacted structure. For example, the nucleic acidnanostructure may be coupled to additional nucleic acid nanostructuresto form a nucleic acid nanostructure complex with an increased surfacearea. In another example, a nucleic acid nanostructure may furthercomprise a pervious structure (e.g., a plurality of pendantoligonucleotides such as shown in FIG. 57 ) that is configured toincrease an effective surface area of the nucleic acid nanostructure. Anucleic acid nanostructure may comprise a compacted structure, in whichthe compacted structure comprises an effective surface area of no morethan about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,or less than 1% of a surface area of an array site. Alternatively oradditionally, a nucleic acid nanostructure may comprise a compactedstructure, in which the compacted structure comprises an effectivesurface area of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more than 90% of a surface area of an array site.

A nucleic acid nanostructure may comprise a pervious region that isconfigured to increase an effective surface area or a footprint of thenucleic acid nanostructure. In some configurations, a nucleic acidnanostructure may comprise a pervious structure, in which the perviousstructure is configured to couple to the site of the solid support(e.g., comprises a capture moiety). In some configurations, a perviousregion may comprise an effective surface area or footprint that islarger than an effective surface area or footprint of a compactedregion. In other configurations, a pervious region may comprise aneffective surface area or footprint that is smaller than an effectivesurface area or footprint of a compacted region.

In some configurations, a nucleic acid nanostructure, when coupled to asolid support, may comprise a total footprint that is larger than atotal effective surface area of the nucleic acid nanostructure when notcoupled to a solid support. A nucleic acid nanostructure, when coupledto a solid support, may comprise a total footprint that is at leastabout 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 110%, 120%, 150%, 200%, or more than 200% of a surface area of anarray site. Alternatively or additionally, a nucleic acid nanostructure,when coupled to a solid support, may comprise a total footprint that isno more than about 200%, 150%, 120%, 110%, 100%, 90%, 80%, 70%, 60%,50%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than 1% of a surface areaof an array site. In some configurations, a nucleic acid nanostructuremay comprise a footprint that exceeds a surface area of an array site.For example, FIG. 56A depicts a nucleic acid nanostructure 5630 withpendant oligonucleotides 5635 (e.g., polynucleotide repeats obtainedfrom TdT extension) whose length in a coupled configuration extendbeyond the array site 5601.

A nucleic acid nanostructure or a component thereof (e.g., a compactedstructure) may comprise a face or profile with a particular shape (e.g.,square, rectangular, triangular, circular, polygonal, etc.). A shape orprofile of a nucleic acid nanostructure or a component thereof may bethe same as, or similar to, the shape or profile of an array site, forexample as determined by an aspect ratio of the shape or profile. Forexample, a square-shaped nucleic acid origami may be coupled to asquare-shaped array site. In a particular configuration, a nucleic acidnanostructure and an array site may comprise the same, or similar, shapeor profile, in which the surface area of the array site is substantiallythe same as a footprint of the nucleic acid nanostructure. In anotherparticular configuration, a nucleic acid nanostructure and an array sitemay comprise the same, or similar, shape or profile, in which a surfacearea of the array site differs from a footprint of the nucleic acidnanostructure (e.g., a larger footprint, a smaller footprint). In otherconfigurations, a shape or profile of a nucleic acid nanostructure or acomponent thereof may comprise a different shape or profile as an arraysite, for example as determined by an aspect ratio of the shape orprofile. For example, a square-shaped nucleic acid origami may becoupled to a circular array site. In some configurations, a shape orprofile of a nucleic acid nanostructure or a component thereof maycomprise a different shape or profile as an array site, in which thenucleic acid nanostructure comprises a larger footprint than a surfacearea of the array site. In other configurations, a shape or profile of anucleic acid nanostructure or a component thereof may comprise adifferent shape or profile as an array site, in which the nucleic acidnanostructure comprises a smaller footprint than a surface area of thearray site. In other configurations, a shape or profile of a nucleicacid nanostructure or a component thereof may comprise a different shapeor profile as an array site, in which the nucleic acid nanostructurecomprises a substantially equal footprint as a surface area of the arraysite.

A plurality of nucleic acid nanostructures or nucleic acid nanostructurecomplexes, as set forth herein, may be combined to form an array. Forexample, a plurality of SNAPs can form a random array (e.g., where theplurality of SNAPS occur in a non-repeating pattern on a surface orinterface) or an ordered array (e.g. where the plurality of SNAPS arespatially arranged in a regular repeating pattern on a surface orinterface). In some configurations, a homogeneous plurality of nucleicacid nanostructures or nucleic acid nanostructure complexes may becombined to form a random or ordered array on a surface or interface. Inother configurations, a heterogeneous plurality of nucleic acidnanostructures or nucleic acid nanostructure complexes may be combinedto form a random or ordered array on a surface or interface. Thehomogeneity or heterogeneity of a plurality of nucleic acidnanostructures or nucleic acid nanostructure complexes may be determinedbased upon the shape, conformation, or structure of the nucleic acidnanostructures or nucleic acid nanostructure complexes. For example, ahomogeneous plurality of nucleic acid nanostructure complexes maycontain only nucleic acid nanostructure complexes with crossconfigurations. In another example, a heterogeneous plurality of nucleicacid nanostructure complexes may contain a mixture of nucleic acidnanostructure complexes with cross or square configurations.

Nucleic acid nanostructures or nucleic acid nanostructure complexes mayarrange at a surface or interface with a characteristic separation orspacing. The characteristic separation or spacing may be determined byan average or localized distance between adjacent analyte coupling siteson differing nucleic acid nanostructures or nucleic acid nanostructurecomplexes. The characteristic separation or spacing may be determinedby: 1) the sizes of nucleic acid nanostructures or nucleic acidnanostructure complexes; 2) the structure or conformation of nucleicacid nanostructures or nucleic acid nanostructure complexes; 3) thespacing or separation of patterning features on a surface; 4) or acombination thereof. For a structured or patterned array, thecharacteristic separation or spacing may be determined by the separationor spacing between structured or patterned features. For an unstructuredor unpatterned array, the characteristic separation or spacing may bedetermined by, for example, the size of nucleic acid nanostructurecomplexes and/or the presence of modifying groups (e.g., steric groups,coupling groups) near the edges of complexes that bind complexestogether or generate inter-complex repulsion. FIGS. 17A-17C depictconfigurations for altering a characteristic separation or spacing of aplurality of nucleic acid nanostructure complexes via the arrangement ofthe complexes. FIG. 17A depicts an assembled array of homogeneous SNAPcomplexes with cross configurations 1710 that are arranged by densepacking of the complexes. The assembled array may have a characteristicspacing between nearest adjacent analyte coupling sites of Δg₁ alongdiagonal lines between coupling sites. FIG. 17B depicts an assembledarray of homogeneous SNAP complexes with cross configurations 1710 thatare arranged with a less dense packing structure than the packing shownin FIG. 17A. The assembled array may have a characteristic spacingbetween nearest adjacent analyte coupling sites of Δg₂ between any twoadjacent analyte coupling sites. FIG. 17C depicts an assembled array ofhomogeneous SNAP complexes with cross configurations 1710 that arecombined with separating SNAPs 1720 (e.g., SNAPs or SNAP complexes withno polypeptide coupling site) to form a separated array. The separatingSNAPs 1720 increase the characteristic spacing Δg₃ between analytecoupling sites on adjacent SNAP complexes. Assuming uniform size of thehomogeneous SNAP complexes with cross configurations 1710, acharacteristic separation of spacing may increase in the orderΔg₃>Δg₂>Δg₁.

In another aspect, provided herein is a single-analyte array comprising:a) a solid support comprising a plurality of addresses, in which eachaddress of the plurality of addresses is resolvable from each otheraddress at single-analyte resolution, and wherein each address isseparated from each adjacent address by one or more interstitialregions; and b) a plurality of analytes, wherein a single analyte of theplurality of analytes is coupled to an address of the plurality ofaddresses, wherein each address of the plurality of addresses comprisesa single analyte of interest (i.e. one and only one analyte ofinterest), wherein each single analyte is coupled to a coupling surfaceof the address by a nucleic acid (e.g., a nucleic acid nanostructure, aSNAP, etc.), and wherein the nucleic acid inhibits (e.g. occludes) thesingle analyte from contacting the coupling surface.

In another aspect, provided herein is a single-analyte array comprising:a) a solid support comprising a plurality of addresses, in which eachaddress of the plurality of addresses is resolvable at single-analyteresolution, in which each address comprises a coupling surface, and inwhich each coupling surface comprises one or more surface-linkedmoieties; and b) a plurality of nucleic acid nanostructures, in whicheach structured nucleic acid particle comprises a coupling moiety, inwhich the coupling moiety comprises a plurality of oligonucleotides, inwhich each oligonucleotide of the plurality of oligonucleotidescomprises a surface-interacting moiety, in which each structured nucleicacid particle of the plurality of structured nucleic acid particles iscoupled to an address of the plurality of addresses by a binding of thesurface-interacting moiety of the plurality of oligonucleotides to asurface-linked moiety of the one or more complementary oligonucleotides,and in which a structured nucleic acid particle of the plurality ofstructured nucleic acid particles comprises a display moiety comprisinga coupling site that is coupled to an analyte.

In some configurations, a single-analyte array may comprise an orderedarray. In particular configurations, a coupling surface of an orderedarray may be formed by a lithographic process. In other particularconfigurations, an address of a plurality of addresses of an orderedarray may be adjacent to one or more interstitial regions, wherein aninterstitial region of the one or more interstitial regions does notcomprise a coupling surface. An interstitial region of one or moreinterstitial regions, as set forth herein, may comprise a disruptingmoiety, in which the disrupting moiety is configured to reduce, prevent,or inhibit a likelihood of a coupling of a molecule (e.g., an affinityreagent, a fluorophore, etc.) to the interstitial region. In someconfigurations, an ordered array may comprise a coupling surface, inwhich the coupling surface comprises a raised surface or a depressedsurface relative to an interstitial region of one or more interstitialregions.

In other configurations, a single-analyte array may comprise anunordered array. An unordered array may comprise a solid support thatdoes not comprise coupling surfaces formed by a patterning process(e.g., lithography). An unordered array may comprise, for example, asubstantially planar solid support comprising a near-uniform surfacelayer comprising a plurality of surface-linked moieties. An unorderedarray may comprise unique, resolvable addresses for nucleic acidnanostructure localization, for example by depositing nucleic acidnanostructures that are configured to prevent co-localization ofmultiple nucleic acid nanostructures at a single address, or bydepositing nucleic acid nanostructures at a concentration that inhibitsco-localization.

In particular configurations, an array, whether ordered or unordered,may further comprise a lipid layer (e.g., a monolayer, bilayer, micelle,or colloid) adjacent to the solid support. A nucleic acid nanostructure(e.g., a SNAP) may be anchored to an array via a lipid bilayer, forexample if a surface-linked moiety of one or more surface-linkedmoieties is coupled to a lipid molecule of the lipid layer. Inparticular configurations, a lipid molecule of a lipid layer maycomprise a phospholipid, triglyceride, or a cholesterol.

In some configurations, a plurality of nucleic acid nanostructures ornucleic acid nanostructure complexes, as set forth herein, may becombined to form a self-assembling or self-patterning array. Analyte maybe conjugated to nucleic acid nanostructures or nucleic acidnanostructure complexes before, during, or after the formation of aself-assembling or self-patterning array to form an array of analyte.Formation of a self-assembling or self-patterning array may be driven byinteractions between nucleic acid nanostructures and a surface orinterface, interactions between nucleic acid nanostructure complexes anda surface or interface, interactions between two or more nucleic acidnanostructures, interactions between two or more nucleic acidnanostructure complexes, or a combination thereof. A self-assembling orself-patterned array of nucleic acid nanostructures or nucleic acidnanostructure complexes may be stable, meta-stable or unstable.Stability and/or order of a self-assembled or self-patterning array ofnucleic acid nanostructures or nucleic acid nanostructure complexes maybe mediated by covalent, non-covalent, electrostatic, or magneticinteractions. For example, a self-assembling array of SNAP complexes maybe stabilized by electrostatic interactions between constituent SNAPsand a surface, plus nucleic acid coupling between adjacent SNAPs. Suchan array may be destabilized by excess temperature or the presence of adenaturant. In another example, a self-assembling array may be formed bycovalent cross-linking between neighboring SNAP complexes that areassociated with a multi-phase interface. The covalently cross-linkedarray may have substantial chemical stability but may be disrupted byexcess mechanical stress.

A self-patterning or self-assembling array of nucleic acidnanostructures or nucleic acid nanostructure complexes may form ahomogeneous or heterogeneous array at a surface or interface. Aself-patterning or self-assembling array of nucleic acid nanostructuresor nucleic acid nanostructure complexes may be homogeneous over anentire surface or interface, or homogeneous over a portion of a surfaceor an interface. FIGS. 18A-18C illustrate array coverage patterns fordiffering configurations of nucleic acid nanostructures or nucleic acidnanostructure complexes in a self-assembling or self-patterning array.FIG. 18A depicts an array of rectangular SNAPs or SNAP complexes 1820that completely occupy a region bordered by box 1810. The ordering orpatterning of the array is approximately homogeneous across the entireregion 1810. FIG. 18B depicts an array of rectangular SNAPs or SNAPcomplexes 1820 that partially occupy a region bordered by box 1810. Thearray is heterogeneous in coverage with respect to region 1810, but isapproximately homogeneous in the subregion bordered by box 1840. Theremaining region 1830 between region 1810 and subregion 1840 may have noSNAPs or SNAP complexes, unorganized or non-arrayed SNAPs or SNAPcomplexes, or smaller arrays of SNAPs or SNAP complexes. FIG. 18Cdepicts an array of rectangular SNAPs or SNAP complexes 1820 that arehomogeneously distributed within a region bordered by box 1810. Thedispersion of SNAPs or SNAP complexes 1820 includes unoccupiedsubregions with few or no SNAPs or SNAP complexes 1830. A homogeneousdispersion with unoccupied subregions may be formed by, for example,depositing SNAPs or SNAP complexes on a patterned array or combining aplurality of SNAPs or SNAP complexes comprising modifying groups thatsterically repel other SNAPs or SNAP complexes.

A plurality of nucleic acid nanostructures or nucleic acid nanostructurecomplexes, as set forth herein, may assemble into a cohesive and/orcontinuous structure. For example, a plurality of nucleic acidnanostructures or nucleic acid nanostructure complexes may form amonolayer or membrane. A cohesive and/or continuous structure may formin a solution then deposit on a surface due to sedimentation or otherdeposition mechanism. A cohesive and/or continuous structure comprisinga plurality of assembled nucleic acid nanostructures or nucleic acidnanostructure complexes may form on a surface or at an interface. FIGS.19A-19B illustrate cohesive or continuous structures formed by theassembly of a plurality of nucleic acid nanostructures or nucleic acidnanostructure complexes. FIG. 19A depicts a plurality of SNAPs 1930 thatare configured to associate with an interface 1950 formed between afirst denser fluid 1960 and a second less dense fluid 1970. Theplurality of SNAPs 1930 are coupled into an analyte array by nucleicacid couplings 1940. The analyte array is further stabilized bycouplings 1920 (e.g. streptavidin-biotin, covalent bonds formed by aclick reaction, etc.) that secure the analyte array to a vessel 1910that contains the first denser fluid 1960 and the second less densefluid 1970. FIG. 19B illustrates a plurality of SNAPs 1930 that arecoupled into an analyte array by nucleic acid couplings 1940. Theanalyte array may form at an interface 1950 or within a fluid 1960before depositing on a surface of a vessel 1910 that contains the fluid1960. Without wishing to be bound by theory, the deposition of theassembled analyte array at the surface may be driven by hydrodynamicdestabilization caused by array size, density, weight, or otherproperties.

A self-patterning or self-assembling array of nucleic acid nanostructurecomplexes may comprise multiple species or configurations of nucleicacid nanostructure complexes, as set forth herein. Species of nucleicacid nanostructure complexes may be distinguished by shape,configuration (e.g., presence or absence of modifying groups, presenceof absence of coupling groups, etc.), presence or absence of aparticular tag, or coupling specificity. Two or more species of nucleicacid nanostructure complexes may be configured to self-assemble intosubregions of a larger array. Two or more species of nucleic acidnanostructure complexes may self-assemble due to complementary couplinggroups (e.g., nucleic acids) on each species of a nucleic acidnanostructure complexes.

Differing species of nucleic acid nanostructures or nucleic acidnanostructure complexes, as set forth herein, may be formed for thepurpose of distinguishing different types of analytes. In someconfigurations, an analyte sample may be divided into separate fractions(e.g., by size, by charge, by mass, by polarity, by location in cell,etc.), with each separate fraction being placed on a different speciesof nucleic acid nanostructure or nucleic acid nanostructure complex. Inother configurations, sample analytes may be coupled to one species ofnucleic acid nanostructure or nucleic acid nanostructure complex and astandard or control analyte may be coupled to a different species ofnucleic acid nanostructure or nucleic acid nanostructure complex. FIG.20 illustrates a method of forming differing species of SNAPs or SNAPcomplexes by selectively targeting polypeptides from a polypeptidesample onto differing SNAPs or SNAP complexes. A square species of SNAPor SNAP complex comprising an amine reactive group 2020 and a triangularspecies of SNAP or SNAP complex comprising a DBCO reactive group 2030are contacted with a polypeptide sample comprising differentiallylabeled polypeptides, including carboxylated polypeptides 2010,activated ester-labeled polypeptides 2011, azide-labeled polypeptides2012, and hydroxyl-labeled polypeptides 2013. Due to the relativereactivities of the SNAP-based reactive groups and the polypeptide-basedreactive groups, the square species of SNAP or SNAP complex 2020covalently conjugates to the activated ester-labeled polypeptide 2011 toform a polypeptide-coupled SNAP or SNAP complex. Likewise, thetriangular species of SNAP or SNAP complex 2030 covalently conjugates tothe activated ester-labeled polypeptide 2012 to form apolypeptide-coupled SNAP or SNAP complex.

Two differing species of nucleic acid nanostructures or nucleic acidnanostructure complexes in an assembled array may be distinguished bydiffering types of displayed analytes. Differing analytes may be sortedon the basis of any analyte property, including, but not limited tosize, weight, length, cellular location (e.g., extracellular, membrane,cytoplasmic, organelle, nuclear, etc.), organism or system of origin(e.g., cell-free synthesis), isoelectric point, hydrodynamic radius,post-translational modification, or any other measurable or observablepolypeptide characteristic. For example, a first species of SNAPs orSNAP complexes in a polypeptide array may comprise polypeptides from apolypeptide-containing sample and a second species of SNAPs or SNAPcomplexes in a polypeptide array may comprise polypeptides from astandard or control sample (i.e., a quality control marker polypeptide,positive control polypeptide, negative control polypeptide, etc.). Inanother example, polypeptides from a first organism may be placed on afirst species of SNAPs or SNAP complexes and polypeptides from a secondorganism may be placed on a second species of SNAPs or SNAP complexes.

Two or more differing species of nucleic acid nanostructures or nucleicacid nanostructure complexes may assemble to form an array with adistinctive, rational, ordered, or segregated arrangement. FIGS. 22-24depict examples of localized patterning of SNAP complexes to generatedifferent array conformations. Differing species of SNAPs or SNAPcomplexes may self-assemble into ordered or patterned arrays.

FIG. 22 depicts an array of SNAPs or SNAP complexes formed by combiningtwo differing species of SNAPs or SNAP complexes that are geometricallymatched and configured to bind to each other to form a symmetricalarray. The square SNAPs or SNAP complexes may self-arrange into regionsof homogeneous SNAPs that are divided by arranged complexes ofsegregating SNAPs or SNAP complexes 2220. The arranged complexes ofsegregating SNAPs or SNAP complexes 2220 may be readily observable ordetectable by some detection methods (e.g., fluorescence microscopy),allowing rapid spatial identification of the locations in an array ofthe segregated square SNAP or SNAP complexes 2210, or the segregatingSNAPs or SNAP complexes 2220. The self-segregation may be promoted byfabricating SNAPs or SNAP complexes with certain utility facescomprising coupling groups that are intended to couple with SNAPs orSNAP complexes of the same species, and other utility faces comprisingcoupling groups that are intended to couple with SNAPs or SNAP complexesof the differing species. The ordered array may also comprise unoccupiedregions of SNAPs or SNAP complexes that are not configured to couple ananalyte 2230. The unoccupied regions or SNAPs or SNAP complexes that arenot configured to couple an analyte 2230 may be used to maintain arraystability and/or facilitate the formation of the array patterning. FIG.24 depicts a similar array to the array depicted in FIG. 22 utilizingseveral species of SNAPs or SNAP complexes. The large square SNAPs orSNAP complexes 2410, small square SNAPs or SNAP complexes 2411, largeright triangular SNAP complexes 2412, and small right triangular SNAPsor SNAP complexes 2413 may be configured to self-segregate intohomogeneous regions of like SNAPs or SNAP complexes. In someconfigurations, the segregating SNAPs or SNAP complexes 2220 or 2420 maybe coupled with standard or control polypeptides (e.g., quality controlpolypeptides, positive control polypeptides, negative controlpolypeptides, etc.) to generate patterned fiducial or gridding lines forimage registration when detecting SNAP arrays, quality control ofprocesses utilizing SNAP arrays, or the like.

FIG. 23A shows an array of SNAPs or SNAP complexes formed by combiningtwo differing species of SNAPs or SNAP complexes that are geometricallymismatched but configured to bind to each other. The binding of ahexagonal SNAP or SNAP complex 2310 to a square SNAP or SNAP complex2320 creates mismatches or discontinuities in the arrangement patternsof arrayed SNAPs or SNAP complexes. The mismatches or discontinuitiesmay be readily observable or detectable by some detection methods (e.g.,fluorescence microscopy), allowing rapid spatial identification of thelocations in an array of the square SNAP or SNAP complexes 2320. Thistype of array may be useful in situations where one species of SNAP orSNAP complex is fewer in total number relative to a second species ofSNAP or SNAP complex. FIG. 23B shows an array of SNAPs or SNAP complexesformed by combining two differing species of SNAPs or SNAP complexesthat are geometrically mismatched but configured to bind to each other.The binding of a hexagonal SNAP or SNAP complex 2310 to a square SNAP orSNAP complex 2320 creates mismatches or discontinuities in thearrangement patterns of arrayed SNAPs or SNAP complexes. In someconfigurations (e.g., approximately equal concentrations of eachspecies), both species may selectively self-segregate, leading tolimited regions of binding between the two species. The locations ofmismatches or discontinuities may be readily observable or detectable bysome detection methods (e.g., fluorescence microscopy), allowing rapidspatial identification of the locations in an array of the segregatedsquare SNAP or SNAP complexes 2320, or the segregated hexagonal SNAPs orSNAP complexes 2310.

An array comprising a plurality of nucleic acid nanostructures ornucleic acid nanostructure complexes, as set forth herein, may remainstable for a particular time period. The stability of an array may be afunction of a threshold quantity of nucleic acid nanostructures ornucleic acid nanostructure complexes remaining coupled to or with thearray. For example, a stable array may comprise at least about 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, ormore than 99% of nucleic acid nanostructures or nucleic acidnanostructure complexes remaining coupled to the array after a setperiod of time such as, for example, at least about 1 s, 1 min, 5 min,10 min, 30 min, 1 hr, 3 hr, 6 hr, 12 hr, 1 day, 2 day, 3 day, 1 week, 1month, 6 months, 1 year, 5 years, or more than 5 years.

In some configurations, a capture moiety of a nucleic acid nanostructuremay be coupled to a coupling surface of a solid support. In otherconfigurations, a capture moiety of a nucleic acid nanostructure neednot be coupled to the surface. For example, a SNAP may be uncoupled(e.g., suspended or solvated in a fluidic medium) from a couplingsurface before deposition of the SNAP, or after the SNAP has beenselectively released from the coupling surface (e.g., via cleavage of acleavable linker). A solid support may comprise any conceivable materialor combinations thereof, including metals, metal oxides, glasses,ceramics, semiconductors, and polymers. A solid support may comprise agel such as a hydrogel. A solid support may comprise a plurality ofsurface-displayed functional groups or moieties (e.g., amines, epoxides,carboxylates, polymer chains, oligonucleotides, etc.). Functional groupsmay be displayed on a solid support, for example, to passivate thesurface, provide coupling sites, or block the binding of molecules tothe surface. Surface-displayed functional groups may be configured toform covalent interactions or non-covalent interactions with a nucleicacid nanostructure (e.g., a SNAP) or other molecule or particle. A solidsupport may further comprise an adjacent or coupled layer, e.g., a lipidmonolayer, a lipid bilayer, a plurality of colloids or micelles, etc. Anadjacent or coupled layer may comprise a plurality of molecules thatalter a surface property of the solid support, such as a surfacetension, a surface energy, a hydrophobicity, a hydrophilicity, or atendency or likelihood to non-specifically bind a particular molecule(e.g., a protein). An adjacent or coupled layer may comprise asurfactant or a detergent species. An adjacent or coupled layer maycomprise a lipid species, such as a phospholipid, a triglyceride, or asterol.

A solid support may comprise an address comprising one or moresurface-linked moieties, in which the address may be resolvable atsingle-analyte resolution. In some configurations, an address maycomprise one or more surfaces, in which the one or more surfaces maycomprise a coupling surface, and in which the coupling surface comprisesthe one or more surface-linked moieties. In particular configurations,one or more surfaces of an address on a solid support may form athree-dimensional structure on the solid support. For example, athree-dimensional structure may comprise a raised structure (e.g., apillar, post, column, dome, pyramid, convex region, etc.) or a wellstructure (e.g., a concave region, channel or well, such as a picowell,nanowell or a microwell).

A coupling surface of a solid support, as set forth herein, may comprisea plurality of surface-linked moieties (e.g., surface-linkedoligonucleotides, surface-linked reactive groups, surface-linkedcoupling groups, etc.). Surface-linked moieties may be covalently ornon-covalently linked to a coupling surface of a solid support. In someconfigurations, a surface-linked moiety distribution or density on acoupling surface may be substantially uniform over the coupling surface.In other configurations, a surface-linked moiety density of a couplingsurface need not be substantially uniform over the coupling surface. Forexample, a fraction of a plurality of surface-linked moieties may belocated within a central region of a coupling surface. In anotherexample, a second fraction of the plurality of surface-linked moietiesmay be located within an outer region of a coupling surface. A pluralityof surface-linked moieties may have an average surface density over aregion of a coupling surface (e.g. the region can be a site or addressof an array) of at least about 0.001 picomoles per square nanometer(pmol/nm²), 0.005 pmol/nm², 0.01 pmol/nm², 0.05 pmol/nm², 0.1 pmol/nm²,0.5 pmol/nm², 1 pmol/nm², 5 pmol/nm², 10 pmol/nm², 50 pmol/nm², 100pmol/nm², or more than 100 pmol/nm². Alternatively or additionally, aplurality of surface-linked moieties may have an average surface densityover a region of a coupling surface of no more than about 100 pmol/nm²,50 pmol/nm², 10 pmol/nm², 5 pmol/nm², 1 pmol/nm², 0.5 pmol/nm², 0.1pmol/nm², 0.05 pmol/nm², 0.01 pmol/nm², 0.005 pmol/nm², 0.001 pmol/nm²,or less than 0.001 pmol/nm².

A solid support, as set forth herein, may comprise a coupling surfacecontaining a plurality of surface-linked moieties, in which a fractionof the surface-linked moieties are coupled to at least onesurface-interacting moiety of a nucleic acid nanostructure (e.g., aSNAP). In some configurations, a fraction of surface-interactingmoieties of a nucleic acid nanostructure is coupled to a fraction ofsurface-linked moieties of a plurality of surface-linked moieties on asolid support. A fraction of surface-interacting moieties coupled to atleast one surface-linked moiety may be at least about 0.000001, 0.00001,0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.25, 0.3, 0.35,0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.99,0.999, 0.9999, 0.99999, or more than 0.99999. Alternatively oradditionally, a fraction of surface-interacting moieties coupled to atleast one surface-linked moiety may be no more than about 0.99999,0.9999, 0.999, 0.99, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55,0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001,0.0005, 0.0001, 0.00001, 0.000001, or less than 0.000001.

FIG. 40C illustrates a configuration of a SNAP composition withdiffering fractions of coupled surface-interacting moieties andsurface-linked moieties. The SNAP 4010 is coupled to the couplingsurface 4002 by binding interactions for each of its 5surface-interacting moieties. Accordingly, the overall fraction ofsurface-interacting moieties coupled to at least one surface-linkedmoiety is 1. The coupling surface comprises a depicted 15 surface-linkedmoieties, of which 5 are involved in forming a binding interaction withthe SNAP 4010. Accordingly, the fraction of surface-linked moietiescoupled to at least one surface-interacting moiety is 0.26. Likewise,fractions can be calculated for each unique type of surface-linkedspecies (e.g., 0.22 for the surface-linked oligonucleotides 4038 and 1for the surface-linked complementary coupling group 4039).

A solid support, as set forth herein, may comprise a passivating layer.A passivating layer may be configured to reduce, inhibit, or preventnon-specific binding of particular molecules (e.g., affinity agents,uncoupled analytes, etc.) with a solid support. In some configurations,a passivating layer may comprise a plurality of molecules that areconfigured to prevent non-specific binding of a molecule to the solidsupport. In particular configurations, a plurality of molecules maycomprise a plurality of surface-linked polymers selected from the groupsconsisting of polyethylene glycol, polyethylene oxide, an alkane, anucleic acid, or a dextran. In some configurations, a molecule of aplurality of molecules comprising a passivating layer may furthercomprise a surface-linked moiety. In some configurations, a passivatinglayer may comprise a molecule of a plurality of molecules that furthercomprises a linker that couples a surface-linked moiety to the couplingsurface. In some configurations, a linking group may comprise a groupthat forms a covalent or coordination bond with a solid support, such asa silane, a phosphate, or a phosphonate.

A random or ordered array of nucleic acid nanostructures or nucleic acidnanostructure complexes may be formed from a plurality of nucleic acidnanostructures or nucleic acid nanostructure complexes at a surface orinterface. A random or ordered array of nucleic acid nanostructures ornucleic acid nanostructure complexes may be formed from a plurality ofnucleic acid nanostructures or nucleic acid nanostructure complexes at astructured or patterned surface. A random or ordered array of nucleicacid nanostructures or nucleic acid nanostructure complexes may beformed from a plurality of nucleic acid nanostructures or nucleic acidnanostructure complexes at an unstructured or non-patterned surface suchas a surface having a continuous lawn or monolith of attachment pointsfor nucleic acid nanostructures or nucleic acid nanostructure complexes.

A structured or patterned surface may be formed on a solid support byany suitable method, such as photolithography, Dip-Pen nanolithography,nanoimprint lithography, nanosphere lithography, nanoball lithography,nanopillar arrays, nanowire lithography, scanning probe lithography,thermochemical lithography, thermal scanning probe lithography, chemicalor plasma etching, local oxidation nanolithography, molecularself-assembly, stencil lithography, or electron-beam lithography. Alithographic method may facilitate formation of a two-dimensional orthree-dimensional feature on a surface of a solid support. In someconfigurations, a substantially planar solid support comprising anoriginal surface may be formed to provide a plurality of sites, in whicheach site of the plurality of sites comprises a face comprising a regionof the original surface, and in which each site of the plurality ofsites is adjacent to one or more interstitial regions, in which the oneor more interstitial regions comprise a formed surface, in which theformed surface comprises a surface produced by a forming process (e.g.,lithography, deposition, etc.). For example, photolithography may beutilized to etch material from a planar solid support, thereby producinga plurality of raised sites surrounded by etched lanes, in which athickness of the solid support at each raised site is substantially thesame as an original thickness of the solid support, and a thickness ofthe solid support at an interstitial region is less than the originalthickness of the solid support. In another example, an array may beformed by patterning a solid material (e.g., a metal, a metal oxide,etc.) onto a surface of a planar solid support to produce a plurality ofsites surrounded by raised interstitial regions of deposited solidmaterial, in which a thickness of the array at each site issubstantially the same as an original thickness of the solid support,and a thickness of the array at an interstitial region is substantiallya sum of the original thickness of the solid support and a thickness ofthe deposited solid material. A site on a solid support may be formedwith a shape or morphology that is substantially the same as a shape ofa nucleic acid nanostructure, as set forth herein. For example, asubstantially square nucleic acid nanostructure may be coupled to asubstantially square array site. A site on a solid support may be formedwith a shape or morphology that is not substantially the same as a shapeof a nucleic acid nanostructure, as set forth herein. For example, asubstantially square nucleic acid nanostructure may be coupled to asubstantially circular array site. In some configurations, a solidsupport, a surface thereof, and/or a site thereof may undergo two ormore surface forming processes to form nanoscale or microscale featureson the surface (e.g. raised features, indented features). For example, asolid support may be formed by photolithography followed by etching(e.g., in potassium hydroxide) to produce a regularly ordered array ofsites, in which each site of the regularly ordered array of sitescomprises a three-dimensional well feature (e.g., a pyramidal well, aconical well, a hemispherical well, etc.). See, for example, Hookway,et. al, Methods, 101, 2016, which is incorporated by reference in itsentirety.

In some configurations, a site of a plurality of sites may comprise athree-dimensional shape or morphology. A forming process (e.g.,lithography) may produce a site or a feature thereof (e.g., a raisedfeature, an indented feature) with a three-dimensional shape ormorphology. FIGS. 66A-66D illustrate particular aspects of sitemorphology for a solid support comprising raised sites, although it willbe readily understood that similar considerations can apply to indentedfeatures or sites. The raised features depicted in FIGS. 66A-66D can beformed by a process that removes material from a solid support or by aprocess that deposits a second solid support material onto a first solidsupport material. FIG. 66A depicts a cross-sectional view of a solidsupport comprising a raised feature (e.g., a site) comprising asubstantially planar top surface 6610 and a lower surface 6612 thatsurrounds the raised feature, in which both surfaces 6610 and 6612 aresubstantially parallel to a bottom surface 6613 of the solid support6613. The raised feature comprises sides surfaces 6611 that aresubstantially orthogonal to the substantially planar top surface 6610and the lower surface 6612, thereby forming a sharp transition 6615 atthe top of the raised feature. The total thickness of the solid support6600 may vary from a maximum thickness, t_(max), between thesubstantially planar top surface 6610 and the bottom surface 6613 to aminimum thickness, t_(min), between the lower surface 6612 and thebottom surface 6613. FIG. 66B depicts a cross-sectional view of a solidsupport comprising a raised feature (e.g., a site) comprising asubstantially planar top surface 6610 and a lower surface 6612 thatsurrounds the raised feature, in which both surfaces 6610 and 6612 aresubstantially parallel to a bottom surface 6613 of the solid support6613. The raised feature comprises side surfaces 6611 that aresubstantially orthogonal to the substantially planar top surface 6610and the lower surface 6612, but the transitions 6616 between the sidesurfaces 6611 and the substantially planar top surface 6610 are diffuse(e.g., rounded, curved, inclined, etc.). The total thickness of thesolid support 6600 may vary from a maximum thickness, t_(max), betweenthe substantially planar top surface 6610 and the bottom surface 6613 toa minimum thickness, t_(min), between the lower surface 6612 and thebottom surface 6613. FIG. 66C depicts a cross-sectional view of a solidsupport comprising a raised feature (e.g., a site) comprising asubstantially planar top surface 6610 and a lower surface 6612 thatsurrounds the raised feature, in which both surfaces 6610 and 6612 aresubstantially parallel to a bottom surface 6613 of the solid support6613. The raised feature comprises sides surfaces 6611 that aresubstantially orthogonal to the substantially planar top surface 6610and the lower surface 6612. The substantially planar top surface 6610comprises one or more non-planar surface features 6617. The non-planarsurface features 6617 may occur due to a natural roughness of a solidsupport material or may be an artifact of an array formation process(e.g., anisotropic lithography, anisotropic deposition of a layer on asurface, anisotropic removal of a processing intermediate such as aphotoresist, etc.). The total thickness of the solid support 6600 mayvary from a maximum thickness, t_(max), between the non-planar surfacefeature 6617 and the bottom surface 6613 to a minimum thickness,t_(min), between the lower surface 6612 and the bottom surface 6613.FIG. 66D depicts a raised feature such as the feature of FIG. 66B, inwhich a plurality of moieties 6620 (e.g., surface-linked moieties) havebeen coupled to the raised feature. Due to the morphology of the surface(e.g., the diffuse transition 6616), orientations of moieties of theplurality of moieties 6620 may vary over the raised feature. In someconfigurations, varied orientations of surface-coupled moieties, forexample near an edge of a site, may facilitate coupling of a nucleicacid nanostructure to a site or a feature thereof. For example, asurface-linked moiety near an edge of an array site may couple to anucleic acid nanostructure adjacent to the array site (e.g., aninterstitial region), thereby permitting re-arrangement of the spatialposition of the nucleic acid nanostructure from the adjacent area to thearray site. In some configurations, varied orientations ofsurface-coupled moieties, for example near an edge of a site, mayinhibit non-specific coupling of entities to a site or a featurethereof. For example, PEG chains near an edge of a site may inhibitbinding of entities (e.g., affinity agents, other nucleic acids) to anarray site when a nucleic acid is already coupled to the array site.

Optionally, a solid support may be formed into an array that isconfigured to couple a plurality of analytes, as set forth herein, by anon-lithographic method. In some cases, an array may comprise a solidsupport comprising a plurality of sites and a separating material, inwhich the separating material separates each site of the plurality ofsites from each other site of the plurality of sites. A separatingmaterial may comprise one or more characteristics of: i) beingconfigured to couple (e.g., covalently couple, non-covalently couple) toa solid support or a surface thereof, ii) providing spatial separationbetween each site of a plurality of sites, iii) facilitating contact ofa nucleic acid nanostructure, as set forth herein, with the solidsupport or the surface thereof, and iv) inhibiting binding of thenucleic acid nanostructure to the separating material. FIG. 64 depictsan array of analytes formed by a non-lithographic method. A solidsupport 6400 may comprise a plurality of nanoparticles or microparticles6410 that arrange on a surface of the solid support 6400 to createspatial regions of the surface of the solid support 6400 that areoccluded from contact with nucleic acids 6420, and wells betweennanoparticles or microparticles 6410 that are sufficiently large enough(e.g., as determined by volume, as determined by area) to facilitatecontact of a nucleic acid 6420 with the surface of the solid support6400. Optionally, the surface of the solid support 6400 may comprise amoiety that facilitates coupling of the nanoparticles or microparticles6410 and/or the nucleic acids 6420. Optionally, a nucleic acid 6420 maybe coupled to an analyte 6430. In some configurations, a separatingmaterial (e.g., a nanoparticle or microparticle) may comprise a surfacecharge (e.g., a carboxylated microparticle, an aminated microparticle)that is configured to form an electrostatic interaction with anelectrically-charged surface moiety (e.g., an amine, a carboxylate,etc.). In particular configurations, a separating material may furthercomprise a passivating moiety that is configured to inhibit binding ofan entity to the separating material (e.g., a PEG moiety, a dextranmoiety, etc.).

An unstructured or non-patterned surface may be formed by any suitablemethod, such as atomic layer deposition, chemical vapor deposition, orchemical liquid deposition. A surface may comprise a plurality offunctional groups to facilitate an interaction with a nucleic acidnanostructure or a nucleic acid nanostructure complex, as set forthherein, such as forming a covalent, non-covalent, or electrostaticinteraction. A surface-bound functional group may include an amine,thiol, carboxylic acid, activate ester, silane, silanol, siloxane,siloxide, silyl halide, silene, silyl hydride, phosphate, phosphonate,epoxide, azide, or sulfhydryl. For example, a silicon-containing surface(e.g., glass, fused silica, silicon wafer, etc.) may comprise amonolayer coating of a silane compound, such as (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl) triethoxysilane (APTES),(3-glycidyloxypropyl) trimethoxysilane (GOPS). FurtherN-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS),11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane,3-iodo-propyltrimethoxysilane, perfluorooctyltrichlorosilane,octylchlorosilane, octadecyltrichlorosilane,(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane, ortridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane. In anotherexample, a metal oxide surface (e.g., ZrO2, TiO₂) may comprise amonolayer of a phosphate or phosphonate compound.

In some configurations, a functional group may comprise a click-typereaction group. In some cases, a functional group may comprise anoligonucleotide. A surface may comprise a passivating layer, such as alayer of PEG, PEO, dextrans, or nucleic acids. A functionalized ornon-functionalized surface may comprise a positive, negative, or neutralelectrical charge.

A solid support or a surface thereof, as set forth herein, may bepatterned to form a patterned or ordered plurality of sites on the solidsupport or surface thereof. A plurality of sites on a solid support or asurface thereof may be considered to be patterned or ordered, forexample, if it comprises one or more characteristics of: i) comprising asubstantially uniform average pitch or average spacing between adjacentsites (e.g., as measured from a center point of a first site to a centerpoint of a second site; as measured from nearest edge of a first site tonearest edge of a second site, etc.), ii) comprising a substantiallyuniform average site size (e.g., as measured by site diameter, sitewidth, site circumference, site surface area, etc.), iii) comprising arepeating pattern of sites or iv) comprising at least a minimum fractionof sites (e.g., at least about 0.8, 0.85, 0.9, 0.95, 0.99, 0.999,0.9999, 0.99999, or more than 0.99999, etc.) in a range comprising theaverage site size between a minimum site size and a maximum site size(e.g., comprising a 0.9 fraction of sites in a diameter range between300 nm and 400 nm). A patterned or ordered grid may comprise a gridgeometry, such as a rectangular grid, a radial grid, or a hexagonalgrid. In some configurations, an array may comprise a plurality ofsites, in which the sites do not conform to a grid or spatial pattern.In particular configurations, a plurality of sites may not conform to agrid or spatial pattern, but the plurality of sites may comprise anaverage pitch and/or average site size that is sufficient forsingle-analyte detection of moieties coupled to a site. A patterned orordered plurality of sites on a solid support or a surface thereof maycomprise one or more sites or addresses that disrupt a pattern,including intentional disruptions (e.g., placement of fiducial elements,placement of separation spaces between subarrays, etc.) andunintentional disruptions (e.g., manufacturing defects, damage, etc.).

A plurality of sites may be characterized as having an averagedisruption rate or an average disruption density. An average disruptionrate may refer to a measured or expected quantity of site disruptionsper a unit quantity of sites (e.g., 1 per 1000, etc.). An averagedisruption density may refer to an areal density of disruptions on asolid support of a surface thereof (e.g., 1 per square centimeter,etc.). As shown in FIG. 63 , a disruption may refer to a site 6310 thathas one or more characteristics of: 1) being misaligned relative to agrid pattern (6321), 2) being a member of a subset of sites that aremisaligned relative to a grid pattern (6324), 3) having a pitch thatfalls below a minimum pitch size (6328), 4) having a pitch that exceedsa maximum pitch size (6327), 5) having a site dimension (e.g., width,length, diameter, area, etc.) that falls below a minimum site dimension(6326), 6) having a site dimension that exceeds a maximum site dimension(6325), 7) comprising an improper morphology (e.g., two-dimensionalshape, three-dimensional topography, etc.) (6322), and 8) lacking astructure (6320, 6323) or chemistry that facilitates moiety deposition.

A solid support or a surface thereof may comprise an average, minimum ormaximum site pitch of at least about 10 nanometers (nm), 50 nm, 100 nm,200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 micron(μm), 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm,1.9 μm, 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm,2.8 μm, 2.9 μm, 3 μm, 3.1 μm, 3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 μm,3.7 μm, 3.8 μm, 3.9 μm, 4 μm, 4.5 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm,50 μm, or more than 50 μm. Alternatively or additionally, a solidsupport or a surface thereof may comprise an average, minimum or maximumsite pitch of no more than about 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5μm, 4.5 μm, 4.0 μm, 3.9 μm, 3.8 μm, 3.7 μm, 3.6 μm, 3.5 μm, 3.4 μm, 3.3μm, 3.2 μm, 3.1 μm, 3.0 μm, 2.9 μm, 2.8 μm, 2.7 μm, 2.6 μm, 2.5 μm, 2.4μm, 2.3 μm, 2.2 μm, 2.1 μm, 2 μm, 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5μm, 1.4 μm, 1.3 μm, 1.2 μm, 1.1 μm, 1 μm, 900 nm, 800 nm, 700 nm, 600nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 10 nm, or less than10 nm. An average pitch may be determined based upon a spatialresolution of a method used to form a solid support (e.g.,photolithography), a desired array density, and/or a necessary spatialseparation between neighboring sites to obtain single-analyte resolutionof moieties bound to each site

A solid support or a surface thereof may comprise an average, minimum ormaximum site size (e.g., width, length, diameter, etc.) of at leastabout 10 nanometers (nm), 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,600 nm, 700 nm, 800 nm, 900 nm, 1 micron (μm),1.1 μm, 1.2 μm, 1.3 μm,1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.1 μm, 2.2 μm,2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, 3 μm, 3.1 μm,3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 μm, 3.7 μm, 3.8 μm, 3.9 μm, 4 μm,4.5 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, or more than 50 μm.Alternatively or additionally, a solid support or a surface thereof maycomprise an average, minimum or maximum site size of no more than 50 μm,40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 4.5 μm, 4.0 μm, 3.9 μm, 3.8 μm, 3.7μm, 3.6 μm, 3.5 μm, 3.4 μm, 3.3 μm, 3.2 μm, 3.1 μm,3.0 μm, 2.9 μm, 2.8μm, 2.7 μm, 2.6 μm, 2.5 μm, 2.4 μm, 2.3 μm, 2.2 μm, 2.1 μm, 2 μm, 1.9μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5 μm, 1.4 μm, 1.3 μm, 1.2 μm, 1.1 μm, 1μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100nm, 50 nm, 10 nm, or less than 10 nm. A site size may be determinedbased upon a spatial resolution of a method used to form a solid support(e.g., photolithography) and/or a size of an analyte or nucleic acidthat is to be deposited on a site.

A binding site or region may have a surface area of at least about 25nm², 100 nm², 500 nm², 1000 nm², 2000 nm², 3000 nm², 4000 nm², 5000 nm²,5500 nm², 6000 nm², 6500 nm², 7000 nm², 7500 nm², 8000 nm², 8500 nm²,9000 nm², 10000 nm², 15000 nm², 20000 nm², 25000 nm², 50000 nm², 100000nm², 250000 nm², 500000 nm², or more than 1000000 nm². Alternatively oradditionally, a binding site or region may have a surface area of nomore than about 1000000 nm², 500000 nm², 250000 nm², 100000 nm², 50000nm², 25000 nm², 20000 nm², 15000 nm², 10000 nm², 9000 nm², 8500 nm²,8000 nm², 7500 nm², 7000 nm², 6500 nm², 6000 nm², 5500 nm², 5000 nm²,4000 nm², 3000 nm², 2000 nm², 1000 nm², 500 nm², 100 nm², 25 nm², orless than 25 nm².

A solid support or a surface thereof, as set forth herein, may comprisea plurality of sites, in which each site of the plurality of sites isconfigured to couple an entity (e.g., an analyte, a nucleic acid, etc.).A solid support, a surface thereof, and/or a site thereof may beprovided with one or more moieties that facilitate a binding interactionwith an entity, such as a nucleic acid. In some configurations, a solidsupport, a surface thereof, and/or a site thereof may be provided withtwo or more differing moieties that facilitate a binding interactionwith an entity, such as a nucleic acid. In some configurations, thefirst moiety of the two or more moieties facilitates a first bindinginteraction and a second moiety of the two or more moieties facilitatesa second binding interaction. In a particular configuration, the firstbinding interaction is the same type of binding interaction as thesecond binding interaction (e.g., both nucleic acid base-pairhybridization, both covalent bonding, both receptor-ligand binding,etc.). In another particular configuration, the first bindinginteraction is a different type of binding interaction from the secondbinding interaction (e.g., a nucleic acid base-pair hybridization and acovalent bonding, a nucleic acid base-pair hybridization and areceptor-ligand binding, etc.). In some configurations, a solid support,a surface thereof, and/or a site thereof may be provided with two ormore differing moieties, in which a first moiety of the two or moremoieties facilitates a first binding interaction with a first bindingaffinity for a first binding complement, and a second moiety of the twoor more moieties facilitates a second binding interaction with a secondbinding affinity for a second binding complement. In a particularconfiguration, a first binding affinity of a first moiety for a firstbinding complement may be stronger than a second binding affinity of asecond moiety for a second binding complement. For example, a surfacemay comprise a mixture of oligonucleotides and streptavidin, in whichthe streptavidin has a significantly stronger affinity for biotin thanthe oligonucleotide has for its complementary oligonucleotide. In otherconfigurations, a first binding affinity of a first moiety for a firstbinding complement may be substantially equal to a second bindingaffinity of a second moiety for a second binding complement. Forexample, a surface may comprise a mixture of a first oligonucleotide anda second oligonucleotide, in which both have substantially similaraffinities for their respective complementary oligonucleotides. In otherconfigurations, a first binding affinity of a first moiety for a firstbinding complement may be stronger than a second binding affinity of asecond moiety for the first binding complement. For example, a surfacemay comprise a mixture of a first oligonucleotide and a secondoligonucleotide, in which sequences of the first and secondoligonucleotides differ by a single nucleotide, and in which the secondnucleotide has a marginally lower affinity for the complementaryoligonucleotide of the first oligonucleotide due to the misalignment ofthe single nucleotide. A binding affinity between a surface moiety and acomplement or ligand may be characterized by a quantitative measure,such as a dissociation constant (K_(D)), an on-rate (k_(on)), or anoff-rate (k_(off)). A binding affinity between a surface moiety and acomplement or ligand may have a dissociation constant of no more thanabout 1 milliMolar, 100 micromolar (μM), 10 μM, 1 μM, 100 nanomolar(nM), 10 nM, 1 nM, 100 picoMolar (pM), 10 pM, 1 pM, 0.1 pM, 0.01 pM, orless than 0.01 pM. Alternatively or additionally, a binding affinitybetween a surface moiety and a complement or ligand may have adissociation constant of at least about 0.01 pM, 0.1 pM, 1 pM, 10 pM,100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, or more than 1mM. In some cases, a solid support, a surface thereof, and/or a sitethereof may comprise a first moiety and a second moiety, in which afirst dissociation constant for a first moiety and its bindingcomplement and a second dissociation constant for a second moiety andits binding complement may differ by at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10 orders of magnitude. In other cases, a solidsupport, a surface thereof, and/or a site thereof may comprise a firstmoiety and a second moiety, in which a first dissociation constant for afirst moiety and its binding complement and a second dissociationconstant for a second moiety and its binding complement may differ by nomore than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than 1 order ofmagnitude.

FIGS. 60A and 60B depict surface chemistry configurations of a solidsupport comprising two or more differing moieties. FIG. 60A depicts asolid support 6000 comprising a site, in which the site 6001 comprises aplurality of oligonucleotides 6010 and a plurality of polymer chains6020 (e.g., PEG chains). The plurality of oligonucleotides 6010 and theplurality of polymer chains 6020 comprise a substantially homogeneousspatial distribution on the site 6001. Optionally, the plurality ofoligonucleotides 6010 and the plurality of polymer chains 6020 maycomprise a heterogeneous spatial distribution on the site 6001. Theconfiguration of FIG. 60A may be useful for coupling a nucleic acidnanostructure (e.g., a SNAP) while preventing non-specific binding of anon-nucleic acid entity (e.g., an analyte). FIG. 60B illustrates a solidsupport 6000 comprising a site, in which the site 6001 comprises aplurality of oligonucleotides 6010, a plurality of polymer chains 6020(e.g., PEG chains), and an additional coupling moiety 6030 (e.g., aClick-type reagent, a streptavidin, etc.). In some configurations, anoligonucleotide of the plurality of oligonucleotides 6010 may asignificantly different binding affinity than the additional couplingmoiety 6030. The configuration of FIG. 60B may be useful for weaklycoupling a nucleic acid nanostructure to a site 6001, then more stronglycoupling the nucleic acid nanostructure once it has found a more stableconfiguration on the site 6001.

A solid support, a surface thereof, or a site thereof comprising a firstplurality of a first surface-coupled moiety (e.g., a coupling moiety, ahigher affinity binding moiety, etc.) and a second plurality of a secondsurface-coupled moiety (e.g., a non-coupling moiety, a lower affinitybinding moiety, etc.) may be configured with an advantageous molar ratioof the first plurality to the second plurality. A first plurality of afirst surface-coupled moiety and a second plurality of a secondsurface-coupled moiety may have a molar ratio of at least about 1:1,1.5:1, 2:1, 3:1, 5:1, 10:1, 20:1, 50:1, 100:1: 1000:1, 10000:1,100000:1, 1000000:1, or more than 1000000:1. Alternatively oradditionally, a first plurality of a first surface-coupled moiety and asecond plurality of a second surface-coupled moiety may have a molarratio of no more than about 1000000:1, 100000:1, 10000:1, 1000:1, 100:1,50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1.5:1, or less than 1.5:1.

A solid support, a surface thereof, and/or a site thereof may beprovided with two or more differing moieties. In some configurations, afirst moiety of the two or more moieties facilitates a first bindinginteraction and a second moiety of the two or more moieties inhibits abinding interaction. For example, a surface of a site may befunctionalized with a first plurality of oligonucleotides that areconfigured to bind complementary oligonucleotides of a nucleic acidnanostructure, and a second plurality of PEG moieties that areconfigured to inhibit non-specific binding of non-nucleic acid entitiesto the surface of the site.

A surface chemistry or functionalization may be provided to a solidsupport, a surface thereof, and/or a site thereof by an appropriatemethod, such as chemical vapor deposition or chemical liquid deposition.A surface chemistry deposition method may include one or more steps toform a layer, or a plurality of layers on a solid support, a surfacethereof, and/or a site thereof. For example, a method of providing aplurality of surface-linked oligonucleotides to a surface may comprisethe steps of: i) coupling a plurality of aminated silane molecules tothe surface, and ii) coupling an azide-terminated PEG molecule to eachsilane molecule, iii) coupling a dibenzocyclooctylene (DBCO)-terminatedoligonucleotide to each azide group. In some configurations, an impurityfrom a surface synthesis may be expected to be present on a solidsupport, a surface thereof, and/or a site thereof. For example, in theprior example of providing a surface layer of oligonucleotides, someunreacted azide may be present on the surface. In some configurations, asurface impurity may be passivated by contacting a passivating moleculewith the surface impurity. A passivating molecule may form a covalentbond with a surface impurity to passivate the impurity. A passivatingmolecule need not form a covalent bond with a surface impurity topassivate the impurity (e.g., an electrostatic interaction). In someconfigurations, a surface impurity may facilitate binding of an entity(e.g., a nucleic acid nanostructure) to a solid support, a surfacethereof, and/or a site thereof.

FIGS. 61A-61E illustrate a method of coupling a nucleic acidnanostructure to a surface. FIG. 61A shows contacting of asilicon-containing surface 6100 with a plurality of silanated moleculescomprising a PEG chain 6110 and a terminal azide group 6111. FIG. 61Billustrates the surface 6100 coupled by covalent bonds 6112 to the PEGchains 6110 with terminal azide groups 6111. The surface is contactedwith a plurality of poly-A oligonucleotides 6120 comprising terminalDBCO moieties that are configured to form a covalent bond with azidegroups 6111. FIG. 61C displays the surface 6100 now comprising PEGchains 6110 terminated with poly-A oligonucleotides 6120, excluding atleast one unreacted azide group 6111. The surface is contacted by anucleic acid nanostructure 6130 comprising a plurality of complementarypoly-T oligonucleotides 6135 and a DBCO moiety 6132. FIG. 61D shows acoupling of the nucleic acid nanostructure 6130 to the surface 6100 dueto the nucleic acid hybridization of the poly-A oligonucleotides 6120 tothe poly-T oligonucleotides 6135 of the nucleic acid nanostructure 6130.FIG. 61E depicts a subsequent step of reacting the DBCO moiety 6132 ofthe nucleic acid nanostructure 6130 to the unreacted azide group 6111 tocovalently bind the nucleic acid nanostructure 6130 to the surface 6100.

A solid support, a surface thereof, and/or a site thereof may beconfigured to form a multiplexed array of analytes. A multiplexed arrayof analytes may comprise a plurality of sites, in which each site of afirst subset of sites of the plurality of sites comprises an analyte ofa first plurality of analytes, and in which each site of a second subsetof sites of the plurality of sites comprises an analyte of a secondplurality of analytes. A multiplexed array may comprise a firstplurality of analyte and a second plurality of analytes, in which thefirst plurality of analytes and the second plurality of analytes differin at least one aspect (e.g., type, source, preparation method, etc.). Amultiplexed array may comprise a first plurality of analyte and a secondplurality of analytes, in which the first plurality of analytes and thesecond plurality of analytes do not differ in at least one aspect (e.g.,duplicate or replicate samples, etc.). In some configurations, a solidsupport that is configured to form a multiplexed array may comprise asubstantially uniform surface chemistry (e.g., solid support compositionand/or composition of surface-coupled moieties on the solid support orsites thereof). For example, FIGS. 50A-50B depict formation of amultiplexed array of analytes, in which a first plurality of analytes5020 and a second plurality of analytes 5025 are coupled to nucleic acidnanostructures 5010, in which the nucleic acid nanostructures 5010 forthe first plurality of analytes 5020 comprise a first functional nucleicacid 5030, and in which the nucleic acid nanostructures 5010 for thesecond plurality of analytes 5020 comprise a second functional nucleicacid 5035. In such an example, the type of analyte coupled to a nucleicacid nanostructure 5010 is configured to be identified based upon thecomponent functional nucleic acid, thereby facilitating use of asubstantially uniform surface chemistry on each site of the array and asubstantially uniform structure of a capture face or capture moiety ofeach nucleic acid nanostructure 5010.

In other configurations, a multiplexed array may comprise a plurality ofsites, in which a first subset of the plurality of sites comprises afirst coupling moiety and a second subset of the plurality of sitescomprises a second coupling moiety, in which the first coupling moietyis configured to couple a first entity (e.g., a nucleic acidnanostructure, an analyte, etc.), and in which the second couplingmoiety is configured to couple a second entity. In a particularconfiguration, the first subset of the plurality of sites comprises aspatially contiguous or spatially consecutive group of sites (e.g., acluster of sites), and/or in which the second subset of the plurality ofsites comprises a spatially contiguous or spatially consecutive group ofsites. In another particular configuration, the first subset of theplurality of sites does not comprise a spatially contiguous or spatiallyconsecutive group of sites (e.g., a cluster of sites), and/or in whichthe second subset of the plurality of sites does not comprise aspatially contiguous or spatially consecutive group of sites.

FIGS. 62A-62E depict methods of forming an array of sites that isconfigured for multiplexing of analytes. FIG. 62A depicts a method ofprinting an array to form two or more regions with differing bindingcharacteristics. In a first step, a solid support 6200 comprising anarray of sites 6201 may be provided. In a second step, a barriermaterial 6210 (e.g., a photoresist) may be provided to portions of thesolid support 6200 to divide a first contiguous subset of sites of theplurality of sites 6201 from a second contiguous subset of sites of theplurality of sites 6201. In a third step, a printing device 6220 (e.g.,an ink-based printer) may deposit a first fluidic medium 6221 comprisinga first species of coupling moiety 6222 in contact with the firstcontiguous subset of sites of the plurality of sites 6201, and maydeposit a second fluidic medium 6225 comprising a second species ofcoupling moiety 6226 in contact with the second contiguous subset ofsites of the plurality of sites 6201. Optionally, after depositing thefirst species of coupling moiety 6222 on the first contiguous subset ofsites of the plurality of sites 6201, and depositing the second speciesof coupling moiety 6226 on the second contiguous subset of sites of theplurality of sites 6201, the barrier material 6210 may be removed orstripped from the solid support 6200. FIG. 62B depicts a method oflithographically forming an array comprising two or more regions withdiffering binding characteristics. In a first step, a solid supportmaterial 6200 comprising a coupled surface layer 6205 (e.g., apassivating layer), and an optional barrier material 6210 (e.g., aphotoresist) may be provided. In a second step, the barrier material6210 and the coupled surface layer 6205 may be patterned to exposeregions of the solid support 6200. In a third step, a printing device6220 (e.g., an ink-based printer) may deposit a first fluidic medium6221 comprising a first species of coupling moiety 6222 in contact witha first contiguous subset of sites of the plurality of sites 6201, andmay deposit a second fluidic medium 6225 comprising a second species ofcoupling moiety 6226 in contact with a second contiguous subset of sitesof the plurality of sites 6201. Optionally, after depositing the firstspecies of coupling moiety 6222 on the first contiguous subset of sitesof the plurality of sites 6201, and depositing the second species ofcoupling moiety 6226 on the second contiguous subset of sites of theplurality of sites 6201, the barrier material 6210 may be removed orstripped from the solid support 6200. FIG. 62C depicts a method oflithographically forming an array comprising randomly distributed. In afirst step, a solid support material 6200 comprising a coupled surfacelayer 6205 (e.g., a passivating layer), and an optional barrier material6210 (e.g., a photoresist) may be provided. In a second step, thebarrier material 6210 and the coupled surface layer 6205 may bepatterned to expose regions of the solid support 6200. In a third step,a printing device 6220 (e.g., an ink-based printer) may deposit afluidic medium 6223 comprising a first species of coupling moiety 6222and a second species of coupling moiety 6226 in contact with theplurality of sites 6201. Optionally, after depositing the first speciesof coupling moiety 6222 and the second species of coupling moiety 6226on the plurality of sites 6201 in a spatially random distribution, thebarrier material 6210 may be removed or stripped from the solid support6200.

FIGS. 62D-62E depict a method of forming a multiplexed array of analytesutilizing an array such as those depicted in FIG. 62A-62C. In a firststep, a solid support comprising a first subset of sites and a secondsubset of sites may be contacted with a first plurality of nucleic acidnanostructures 6241 and a second plurality of nucleic acidnanostructures 6242, in which the first subset of sites comprises afirst coupling moiety 6222 and the second subset of sites comprises asecond coupling moiety 6226, in which each nucleic acid nanostructure ofthe first plurality of nucleic acid nanostructures 6241 is configured tocouple to a first coupling moiety 6222, and in which each nucleic acidnanostructure of the second plurality of nucleic acid nanostructures6242 is configured to couple to a second coupling moiety 6226.Optionally, each nucleic acid nanostructure of the first plurality ofnucleic acid nanostructures 6241 may be coupled to an analyte of a firstplurality of analytes 6251, and each nucleic acid nanostructure of thesecond plurality of nucleic acid nanostructures 6242 may be coupled toan analyte of a second plurality of analytes 6252. In a second step, asingle nucleic acid nanostructure of the first plurality of nucleic acidnanostructures 6241 may deposit at a site comprising a first couplingmoiety 6222, and a single nucleic acid nanostructure of the secondplurality of nucleic acid nanostructures 6242 may deposit at a sitecomprising a second coupling moiety 6226.

A solid support, a surface thereof, and/or a site thereof may beconfigured to couple a nucleic acid nanostructure by a charge-mediatedinteraction. A charge-mediated interaction may be a binding interactionin which an electrically-charged intermediate facilitates an entity(e.g., an analyte, a nucleic acid nanostructure, etc.) in forming abinding interaction with a solid support, a surface thereof, and/or asite thereof. In some configurations, a charge-mediated interaction maycomprise an ion-mediated interaction, in which an ionic species (e.g., acation, an anion) facilitates a coupling interaction between an entityand a solid support, a surface thereof, or a site thereof. For example,a cationic species (e.g., Na⁺, Mg²⁺, Ca²⁺, etc.) may provide anelectrostatic bridging interaction that facilitates binding of a nucleicacid to an electrically-charge surface. In particular configurations, anion-mediated interaction may facilitate a coupling interaction betweenan electrically-charged capture face or capture moiety of a nucleic acidnanostructure and an electrically-charged surface (e.g., a surfacefunctionalized with an amine or carboxylate, etc.), in which theelectrically-charged capture face or capture moiety of the nucleic acidnanostructure and the electrically-charged surface comprise a samepolarity of electrical charge (e.g., both positively charged, bothnegatively charged). For example, magnesium ions may form a bridginginteraction between a negatively-charged nucleic acid and anegatively-charged surface. In another particular configuration, anion-mediated interaction may facilitate a coupling interaction betweenan electrically-charged capture face or capture moiety of a nucleic acidnanostructure and an electrically-charged surface (e.g., a surfacefunctionalized with an amine or carboxylate, etc.), in which theelectrically-charged capture face or capture moiety of the nucleic acidnanostructure and the electrically-charged surface comprise a differingpolarity of electrical charge (e.g., one positively charged, onenegatively charged). For example, a concentration of a cationic speciesor anionic species may be varied to modulate a strength of interactionbetween a positively-charge surface and a negatively-charged nucleicacid.

In some configurations, a charge-mediated interaction may be utilized toform an array of analytes. FIG. 65 depicts a method of forming an arrayof analytes on an unpatterned surface comprising an electrically-chargedspecies. In a first step, a solid support 6500 comprising a plurality ofsurface-coupled, positively-charged species 6510 (e.g., aminatedsilanes) is provided. The solid support 6500 is contacted with aplurality of negatively-charged nanoparticles or microparticles 6520(e.g., carboxylated dextran, carboxylated polystyrene, etc.). Theplurality of negatively-charged nanoparticles or microparticles 6520 maycouple to the surface-coupled, positively-charged species 6510 due to anelectrostatic interaction. In a second step, the formed layer comprisingthe plurality of negatively-charged nanoparticles or microparticles 6520may be contacted with a plurality of nucleic acid nanostructures 6530,as set forth herein. A nucleic acid nanostructure of the plurality ofnucleic acid nanostructures 6530 may be coupled to an analyte 6534. Anucleic acid nanostructure may comprise a capture face or capture moiety(e.g., an amine) comprising a positively-charge moiety that isconfigured to form an electrostatic interaction with anegatively-charged nanoparticle or microparticle 6520. A nucleic acidnanostructure of the plurality of nucleic acid nanostructures 6530 mayfurther comprise a utility face or utility moiety comprising a moiety6532 that is configured to inhibit contact between adjacent nucleic acidnanostructures 6350. In a third step, the plurality of nucleic acidnanostructures 6530 may be deposited on the array, in which each nucleicacid nanostructure 6530 is spatially separated from each adjacentnucleic acid nanostructure 6530, optionally by a utility moiety 6532. Inan optional final step, the electrostatically-coupled array of nucleicacid nanostructures 6530 and negatively-charged nanoparticles ormicroparticles 6520 may be covalently coupled by a cross-linking agent,such as sulfo-N-hydroxysuccinimide (sulfo-NHS), thereby permanentlyconfining the spatial location of each nucleic acid and/or analyte ofthe array.

A solid support, a surface thereof, and/or a site thereof, may beconfigured to form a weak binding interaction with an entity (e.g., ananalyte, a nucleic acid nanostructure, a non-nucleic acid, a reagent).In some configurations, a solid support, a surface thereof, and/or asite thereof, may be configured to form a plurality of weak bindinginteractions with a nucleic acid nanostructure in an initialconfiguration, and in which the solid support, the surface thereof,and/or the site thereof, is configured to facilitate a rearrangement ofthe nucleic acid nanostructure from the initial configuration to amore-stable final configuration. Without wishing to be bound by theory,a weak binding interaction may comprise a coupling of a first moiety(e.g., a surface-coupled moiety) to a second moiety (e.g., a capturemoiety), in which the weak binding interaction is weakly biased towardassociation or dissociation (e.g., an equilibrium constant between about0.01 and 100, about 0.05 and 50, about 0.1 and 10, about 0.5 and 5,etc.), and/or in which the weak binding interaction is kineticallyreversible on a time-scale shorter than a time-scale of an array-basedprocess (e.g., capable of dissociating within a time-scale of a nucleicacid deposition process, capable of dissociating during an array rinsingprocess, etc.).

A plurality of moieties may be provided to a solid support, a surfacethereof, and/or a site thereof, in which a subset of the plurality ofmoieties is configured to form a plurality of binding interactions withone or more surface-coupling moieties of a nucleic acid nanostructure.In some configurations, a subset of a plurality of moieties may coupleto one or more coupling moieties of a nucleic acid nanostructure,thereby coupling the nucleic acid nanostructure to a solid support, asurface thereof, and/or a site thereof. In a particular configuration, asolid support, a surface thereof, and/or a site thereof may be provideda plurality of moieties, in which the plurality of moieties comprises anexcess of coupling moieties relative to an available quantity of capturemoieties of a nucleic acid nanostructure. For example, a nucleic acidnanostructure may comprise 20 pendant surface-coupling moieties, eachcomprising 10 segmented poly-T repeats of 20 nucleotides length (e.g.,200 total capture moieties), and a site on a solid support may comprise1000 surface-linked poly-A oligonucleotides of 20 nucleotide lengths,thereby giving an excess of 5:1 for surface-linked moieties. In someconfigurations, a solid support, a surface thereof, and/or a sitethereof may comprise a plurality of moieties, in which a subset of theplurality of moieties are not configured to couple to an entity. Forexample, an array site may comprise a first plurality of moietiescomprising oligonucleotides that are configured to couple acomplementary oligonucleotide of a nucleic acid nanostructure and asecond plurality of moieties comprising polymer chains that areconfigured to inhibit non-specific binding interactions between entitiesand the solid support, the surface thereof, and/or the site thereof.

FIGS. 60A-60D present configurations of pluralities of moieties on anarray site that facilitate formation of a plurality of weak bindinginteractions. FIGS. 60A and 60B comprise variations of differing bindingand non-binding moieties, as described herein. FIG. 60C illustrates asolid support 6000 comprising a site 6001 that contains a coupledplurality of moieties, including a first plurality of oligonucleotides6010 that are complementary to a surface-coupling oligonucleotide of anucleic acid nanostructure, a second plurality of oligonucleotides 6011that contain random nucleotide substitutions, thereby providing aplurality of nucleotide sequences with lower binding affinities to thecomplementary surface-coupling oligonucleotides of the nucleic acidnanostructure, and a third plurality of non-binding moieties 6020 (e.g.polymer chains). Such a configuration may be modified to comprise, forexample, a component of a receptor-ligand pair and a modified versionthereof. For example, a surface may be provided an antibody fragment andone or more mutated versions thereof, in which the mutated versions havea lower binding affinity for a ligand of the antibody fragment that iscoupled to a capture face of a nucleic acid nanostructure. FIG. 60Dcomprises a modification of the array site of FIG. 60B, in which theadditional coupling moiety 6030 is effectively buried or screenedamongst other moieties, thereby inhibiting its ability to form bindinginteractions with a complementary coupling moieties of a nucleic acidnanostructure. Such a configuration may be useful for slowing a rate ofinteraction formation for a high-affinity binding system (e.g., aClick-type reaction, streptavidin-biotin, etc.). It may be advantageousto form high-affinity interactions between nucleic acid nanostructuresand solid supports to prevent dissociation of the nucleic acidnanostructure from the solid support, but at a slow enough rate tofacilitate rearrangement of nucleic acid nanostructures into more-stableconfigurations on the solid support and/or facilitate disruption ofco-localized pairs of nucleic acid nanostructures from an address of asolid support before both become permanently coupled to the surface by ahigh-affinity binding interaction. For example, a streptavidin moietymay be buried within a plurality of polymer chains (e.g., PEG, alkanes,dextrans, etc.), thereby necessitating transfer of a complementarybiotin moiety coupled to a nucleic acid nanostructure (e.g., via apolymer linking moiety) through the plurality of polymer chains to thestreptavidin moiety (e.g., by a diffusional mechanism or reptation,etc.).

A surface or solid support, as set forth herein, may comprise a materialwith desired characteristics such as hydrophobicity or hydrophilicity,amphipathicity, low adhesion of particular chemical or biologicalspecies, and particular chemical, optical, electrical, or mechanicalproperties. In some cases, a surface or solid support material may bechosen for its compatibility with a detection technique or method (e.g.,confocal fluorescent microscopy). For example, a material may beselected due to its low autofluorescence characteristic. A surface orsolid support may comprise a solid surface to which molecules can becovalently or non-covalently attached. Non-limiting examples of solidsubstrates include slides, surfaces of elements of devices, membranes,flow cells, wells, chambers, and microfluidic or microfluidic chambers.Surfaces and/or solid supports used herein may be flat or curved, or canhave other shapes, and can be smooth or textured. In some cases, solidsupport surfaces may contain microwells. In some cases, solid supportsurfaces may contain nanowells. Such wells can be configured as sites oraddresses of an array. In some cases, solid support surfaces may containone or more microwells in combination with one or more nanowells, forexample, each microwell accommodating an array of nanowells.

A surface or solid support may comprise polymers, glasses,semiconductors (e.g., silicon, germanium), ceramics, metals, minerals(e.g., mica), or other materials. In some instances, a surface or solidsupport may comprise components made of a glass such as borosilicateglass, fused silica, or quartz. In other instances, a surface or solidsupport may comprise an optical glass or a photochromatic glass. In somecases, a glass with a high sodium or potassium content may be selectedas a material for a fluidic device component. A surface or solid supportmay be fabricated from polymers or plastics such as polycarbonate,polyethylene, polypropylene, polyethylene terephthalate, polyvinylchloride, polymethyl methacrylate, polydimethylsiloxane, polystyreneacrylics, latex and others. A surface or solid support may comprisemetals, metal alloys, metal oxides, metal nitrides, or combinationsthereof, such as stainless steel, gold, chromium, titanium, titaniumoxide, tin oxide, zirconium oxide or aluminum. A surface or solidsupport may comprise carbohydrates such as dextrans or cellulose. Insome cases, a surface or solid support may comprise two or morecomponents with different (e.g. plastic vs. glass) or differing (e.g.borosilicate vs. quartz glass) material types.

A surface or solid support, as set forth herein, may be characterized bya thickness or depth. The thickness of a surface or solid support may beuniform or may vary over the body of the surface or solid support. Thethickness of the surface or solid support may be altered by afabrication, forming or machining process. In some cases, a surface orsolid support may have a thickness of at least about 1 nanometer (nm),10 nm, 100 nm, 1 micrometer (μm), 10 μm, 50 μm, 100 μm, 250 μm, 500 μm,750 μm, 1 millimeter (mm), 5 mm, 1 centimeter (cm), 10 cm or more than10 cm. Alternatively or additionally, a surface or solid support mayhave a thickness of no more than about 10 cm, 1 cm, 5 mm, 1 mm, 750 μm,500 μm, 250 μm, 100 μm, 50 μm, 10 μm, 1 μm, 100 nm, 10 nm, 1 nm, or lessthan 1 nm.

A surface or solid support, as set forth herein, may comprise one ormore surface coatings. A surface coating may be organic or inorganic. Insome cases, a surface coating may be deposited by a suitable depositionprocess, e.g., atomic layer deposition, chemical vapor deposition,chemical liquid deposition, spin coating, self-assembling monolayers. Insome cases, a surface coating may be patterned by a suitable patterningprocess, e.g., dry etch, wet etch, lift-off, deep UV lithography orcombination thereof. A deposited surface coating may have a uniformthickness or a variable thickness over a surface of a solid support. Insome cases, a surface coating may comprise an atomic or molecularmonolayer. In some cases, a surface coating may comprise aself-assembled monolayer or sub-monolayer. In some cases, a surfacecoating may comprise a metal or metal oxide layer. In some cases, asurface coating may comprise a silane layer (e.g., ethoxy-, methoxy- orchloro-silane, silanol, siloxane, etc.), a phosphonate layer, acarboxylate layer (e.g., carboxylate transition metal oxides), a thiollayer (e.g., thiolated gold), or a phosphate layer. In some cases, asurface coating may comprise a polymer, a mineral, a ceramic, or an ink.A surface or solid support may comprise a layer or coating comprising afunctional group or moiety that is configured to couple to acomplementary functional group or moiety on a SNAP or SNAP complex. Asurface or solid support may have a gel coating.

A surface coating on a surface or solid support, as set forth herein,may be characterized by a particular thickness. A surface coating may beat least about 1 Angstrom (Å), 5 Å, 1 nanometer (nm), 5 nm, 10 nm, 20nm, 30 nm, 40 nm, 50 nm, 100 nm, 250 nm, 500 nm, 1 micrometer (μm), 5μm, 10 μm, 50 μm, 100 μm or more. Alternatively or additionally, asurface coating may be no more than about 100 μm, 50 μm, 10 μm, 5 μm, 1μm, 500 nm, 250 nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, 1nm, 5 Å, 1 Å or less.

A surface or a surface coating of a solid support, as set forth herein,may be characterized by a surface roughness. A surface roughness may bedue to an intrinsic character of a material or processing method used toform the material or surface. A surface roughness may be calculated asan average size of roughness features (e.g., depressions, bumps, etc.)or may be provided as a distribution of feature sizes relative to a meanor average surface height or level. A surface may be provided with acoating or layer to alter the average surface roughness or distributionof roughness features on the surface. For example, a surface may becoated to decrease the average surface roughness of a material. In othercases, a surface may be etched, coated, or otherwise treated to increasethe surface roughness.

In some configurations, a SNAP or a SNAP complex may comprise a captureface or capture moiety that is structured to facilitate coupling to asurface with surface roughness. For example, a SNAP may comprise acapture face comprising a plurality of single-stranded nucleic acids orother interacting groups (e.g., electrically-charged moieties, magneticmoieties, etc.) that may form an increased interaction area with asurface. FIG. 25A-25C illustrate examples of forming interactions withsurfaces comprising a surface roughness. FIG. 25A depicts the contactingof a SNAP complex 2510 with component SNAPs having unmodified capturefaces with a surface 2500 comprising surface roughness. The SNAP complex2510 can only form limited interactions with the surface where thecapture faces contact the surface 2500 high points. FIG. 25B depicts thecontacting of a SNAP complex 2510 with component SNAPs having capturefaces modified with single stranded nucleic acids 2520 (or otherinteracting groups) with a surface 2500 comprising surface roughness.The SNAP complex 2510 can form increased interactions with the surfacewhere the single-stranded nucleic acids 2520 contact the surface 2500high points. FIG. 25C illustrates contacting a plurality of SNAPcomplexes 2510 with a nanostructured surface 2500 comprising a pluralityof pillar-type structures 2530. The SNAP complex 2510 may be configuredto facilitate the display of an analyte at the top of eachnanostructured feature of the surface 2500. For example, utility SNAPsof a SNAP complex 2510 may comprise utility moieties (e.g., hydrophobicmoieties) on a utility face that can interact with utility moieties ofother SNAP complexes 2510, thereby increasing the likelihood that theutility SNAPs of adjacent SNAP complexes 2510 co-locate in interstitialregions between raised features and display SNAPs of each SNAP complexbind to the top of a pillar-type structure 2530.

A surface such as a solid support may comprise a characterizedroughness. Surface roughness may be characterized by a method such assurface profilometry, contact profilometry, atomic force microscopy,optical microscopy, or any other suitable technique. A surface maycomprise a characterized average roughness of at least about 0.1 nm, 0.2nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm,3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 m, 13 nm, 14nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, or more than 20 nm. Asurface may comprise a characterized average roughness of no more thanabout 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.9 nm,0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, 0.1 nm, or lessthan 0.1 nm.

A surface or solid support may comprise one or more surfaces that arecoated with a layer of metal or metal oxide. A metal or metal oxidelayer may comprise a particular species depending upon the preferablechemistry. Candidate metals or metal oxides may include zirconium oxide(ZrO₂), hafnium (Hf), gold (Au), titanium dioxide (TiO₂), aluminum (Al),aluminum oxide (Al₂O₃) or a combination thereof.

In some cases, the surface or solid support may be optically opaque. Insome cases, all or part of the solid surface or solid support may beoptically opaque at one or more wavelengths such as the infrared,visible, red, orange, yellow, green, blue, violet or ultraviolet. Insome cases, all or part of the solid surface or solid support may beoptically clear, or may be optically clear at one or more wavelengthssuch as the infrared, visible, red, orange, yellow, green, blue, violetor ultraviolet. For example, a solid surface or solid support may beoptically opaque in regions that are not functionalized, and opticallyclear in regions that are functionalized.

Methods of Coupling Nucleic Acids at Solid Supports

In another aspect, provided herein is a method of coupling a nucleicacid nanostructure to an array site, comprising: a) contacting an arraycomprising a site with a nucleic acid nanostructure, in which the sitecomprises a plurality of surface-linked moieties, and in which thenucleic acid nanostructure comprises a plurality of capture moieties, b)coupling the nucleic acid nanostructure to the site in an initialconfiguration, in which the initial configuration does not comprise astable configuration, and in which the nucleic acid nanostructure iscoupled by a coupling of a capture moiety of the plurality of capturemoieties to a surface-linked moiety of the plurality of surface-linkedmoieties, c) uncoupling the coupling of the capture moiety of theplurality of capture moieties to the surface-linked moiety of theplurality of surface-linked moieties, and d) altering the nucleic acidnanostructure from the initial configuration to the stableconfiguration, in which each capture moiety of the plurality of capturemoieties is coupled to a surface-linked moiety of the plurality ofsurface-linked moieties. Optionally, the nucleic acid nanostructure canbe conjugated to, or configured to conjugate to, an analyte of interest.Other optional compositions for the nucleic acid nanostructure are setforth elsewhere herein.

In some configurations, uncoupling of a capture moiety of a nucleic acidnanostructure from a surface-linked moiety of an array site comprisesheating the solid support and/or the nucleic acid nanostructure, and/orcontacting the solid support with a fluidic medium that is configured touncouple the surface-linked moiety from the capture moiety.

In some configurations, a method of coupling a nucleic acidnanostructure to an array site may comprise contacting the array with afluidic medium, as set forth herein, in which the fluidic mediumcomprises the nucleic acid nanostructure. Optionally, the fluidic mediumcan include a plurality of nucleic acid nanostructures, at least asubset of which couple individually to respective sites of the array. Inparticular configurations, altering a nucleic acid nanostructure from aninitial configuration to a stable configuration may further comprisealtering a fluidic medium that is contacted with a solid support. Insome configurations, altering a fluidic medium in contact with a solidsupport may comprise introducing a chemical species (e.g., a surfactant,a denaturant, a chaotrope, an ionic species, an acid, a base, etc.). Inother configurations, altering a fluidic medium in contact with a solidsupport may comprise altering a concentration of a chemical species inthe fluidic medium (e.g., a surfactant, a denaturant, a chaotrope, anionic species, an acid, a base, etc.).

A method of coupling a nucleic acid nanostructure to an array site mayutilize a nucleic acid nanostructure that comprises one or more capturemoieties that are configured to form a multi-valent binding interaction(e.g., coupling to more than one surface-linked moiety). A capturemoiety of a nucleic acid nanostructure may comprise a structure thatfacilitates formation of a multi-valent binding interaction (e.g., apolynucleotide repeat, a first and second polynucleotide repeatseparated by an intermediate nucleotide sequence, etc.). A capturemoiety may optionally comprise a structure that weakens a bindingstrength or binding specificity of any individual binding interaction ofa multi-valent binding interaction. In some configurations, a nucleicacid nanostructure may comprise a capture moiety comprising ahomopolymer sequence or other composition set forth elsewhere herein,for example, in the context of pendant oligonucleotides and stapleoligonucleotides. The nucleic acid nanostructure may be coupled to asolid support comprising a first surface-linked moiety that iscomplementary to or reactive with the surface coupling moiety. In someconfigurations, a nucleic acid nanostructure may comprise a capturemoiety comprising a nucleotide sequence that containsself-complementarity. A method of coupling a nucleic acid nanostructureto a surface may comprise one or more steps of: i) disrupting aself-complementary nucleotide sequence of a capture moiety, and ii)coupling a surface-linked moiety to the self-complementary nucleotidesequence of the capture moiety (e.g., via a toehold-mediated stranddisplacement reaction, etc.).

A method of coupling a nucleic acid nanostructure to a surface maycomprise: i) coupling the nucleic acid nanostructure to the surface inan initial configuration, and ii) altering the nucleic acidnanostructure to a final configuration, in which the final configurationis more stable (temporally, spatially, thermodynamically, kinetically,etc.) than the initial configuration. In some cases, an initialconfiguration may comprise a spatial positioning of a nucleic acidnanostructure on a site of a solid support, in which the initialconfiguration comprises a non-maximized or partial quantity of couplingsof capture moieties to surface-linked moieties. For example, a nucleicacid nanostructure containing 20 capture moieties may have anon-maximized or partial quantity of coupling if less than 20 of thecapture moieties are coupled to surface-linked moieties of an arraysite. In another example, a nucleic acid nanostructure containing 20capture moieties may be expected to form coupling interaction with atleast 10 surface-linked moieties (e.g., at least 50% of availablebinding groups utilized) to achieve a maximized quantity of coupling. Inother cases, an initial configuration may comprise a non-maximizedfootprint of a nucleic acid nanostructure on an array site. For example,if only a fraction of a nucleic acid nanostructure is coupled to asurface of an array site (see FIG. 58B), then the nucleic acidnanostructure has not maximized its footprint on the array site and mayhave a non-maximized quantity of coupling interactions formed. In othercases, an initial configuration may comprise an asymmetric alignment ofthe nucleic acid nanostructure on the site. For example, a substantiallysquare nucleic acid nanostructure may initially couple to asubstantially square array site, in which a center point of the nucleicacid nanostructure is not aligned with a center point of the array site.In some configurations, a more-stable final configuration may comprise alocation on an array site in which the nucleic acid nanostructure formsa maximized quantity of couplings of capture moieties to surface-linkedmoieties. In other configurations, a more-stable final configuration maycomprise a maximized footprint of the nucleic acid nanostructure on thesite. In other configurations, a more-stable final configuration maycomprise a symmetric alignment of the nucleic acid nanostructure on thesite.

A nucleic acid nanostructure may be coupled to an array site by acoupling of one or more capture moieties of the nucleic acidnanostructure to a plurality of surface-linked moieties of the arraysite. An array site may have an excess quantity of surface-linkedmoieties, in which the excess quantity is determined with respect to aquantity of available binding groups on one or more capture moietiesand/or with respect to a spatial density of available binding groups onthe one or more capture moieties. For example, a nucleic acidnanostructure may comprise 20 capture moieties comprising poly-Tsequences, in which each capture moiety is configured to form about 10binding interactions with surface-linked poly-A oligonucleotides. Insuch a case, an array sites containing more than 200 surface-linkedpoly-A oligonucleotides may be considered to contain an excess quantityof surface-linked moieties. In another example, a nucleic acidnanostructure may comprise a plurality of capture moieties with anaverage surface density of about 1 capture moiety per 10 squarenanometers. In such a case, an array site comprising surface-linkedmoieties with a surface density exceeding 1 surface-linked moiety per 10square nanometers may contain excess quantity of surface-linkedmoieties. An array site may contain a molar excess of surface-linkedmoieties relative to the quantity of available capture moieties of anucleic acid nanostructure, on an absolute or spatial density basis, ofat least about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold,1000-fold, 5000-fold, 10000-fold, 100000-fold, 1000000-fold, or morethan 1000000-fold. Alternatively or additionally, an array site maycontain a molar excess of surface-linked moieties relative to thequantity of available capture moieties of a nucleic acid nanostructure,on an absolute or spatial density basis, of no more than about1000000-fold, 100000-fold, 10000-fold, 5000-fold, 1000-fold, 500-fold,250-fold, 100-fold, 50-fold, 25-fold, 10-fold, 5-fold, 4-fold, 3-fold,2-fold, 1.5-fold, 1.2-fold, 1.1-fold, or less than 1.1 fold. In otherconfigurations, an array site may comprise a molar deficit ofsurface-linked moieties relative to the quantity of available capturemoieties of a nucleic acid nanostructure.

Provided herein is a method of forming an array, comprising providing aplurality of nucleic acid nanostructures or nucleic acid nanostructurecomplexes, as set forth herein, coupling each nucleic acid nanostructureor nucleic acid nanostructure complex of the plurality of nucleic acidnanostructures or nucleic acid nanostructure complexes to one or moreadditional nucleic acid nanostructures or nucleic acid nanostructurecomplexes from the plurality of nucleic acid nanostructures or nucleicacid nanostructure complexes, and coupling each nucleic acidnanostructure or nucleic acid nanostructure complex of the plurality ofnucleic acid nanostructures or nucleic acid nanostructure complexes witha surface, where each nucleic acid nanostructure or nucleic acidnanostructure complex comprises a display nucleic acid nanostructure andone or more capture nucleic acid nanostructures or utility nucleic acidnanostructures, and wherein each nucleic acid nanostructure complexcomprises a coupling moiety that couples with the surface, therebyforming an array.

In some configurations, each nucleic acid nanostructure complex isassembled prior to being contacted with another nucleic acidnanostructure complex to which it will couple. In other configurations,individual nucleic acid nanostructures are contacted with each other toresult in conjugation of nucleic acid nanostructure complexes to othernucleic acid nanostructure complexes. Accordingly, a method of formingan array, can include providing a plurality of nucleic acidnanostructures to produce a plurality of nucleic acid nanostructurecomplexes, each nucleic acid nanostructure complex comprising at leasttwo nucleic acid nanostructure complexes that are coupled together, andcoupling the plurality of nucleic acid nanostructure complexes with asurface, where each nucleic acid nanostructure complex comprises adisplay nucleic acid nanostructure and one or more utility nucleic acidnanostructures, and where each nucleic acid nanostructure complexcomprises a coupling moiety that couples with the surface, therebyforming an array.

A display nucleic acid nanostructure may be coupled to an analyte beforeor after being incorporated into an array. In some configurations, amethod may further comprise a step of coupling an analyte to the displaymoiety. In some configurations, an analyte may be coupled to a displaymoiety after a coupling of each nucleic acid nanostructure complex of aplurality of nucleic acid nanostructure complexes with a surface. Insome configurations, an analyte may be coupled to a display moietybefore a coupling of each nucleic acid nanostructure complex of aplurality of nucleic acid nanostructure complexes with a surface. Insome configurations, an analyte may be coupled to a display moiety aftera coupling of each nucleic acid nanostructure complex of a plurality ofnucleic acid nanostructure complexes to one or more additional nucleicacid nanostructure complexes from a plurality of nucleic acidnanostructure complexes. In some configurations, an analyte may becoupled to a display moiety before a coupling of each nucleic acidnanostructure complex of a plurality of nucleic acid nanostructurecomplexes to one or more additional nucleic acid nanostructure complexesfrom a plurality of nucleic acid nanostructure complexes. In someconfigurations, an analyte may be coupled to a display moiety after aproviding of a plurality of nucleic acid nanostructure complexes. Insome configurations, an analyte may be coupled to a display moietybefore a providing of a plurality of nucleic acid nanostructurecomplexes.

An array comprising a nucleic acid nanostructure or a nucleic acidnanostructure complex, as set forth herein, may be formed in aparticular formation condition. A condition may include a particularsolvent or buffering condition. In some configurations, a plurality ofnucleic acid nanostructures or nucleic acid nanostructure complexes maybe provided in a pH buffer comprising a magnesium salt. In someconfigurations, coupling of a plurality of nucleic acid nanostructuresor nucleic acid nanostructure complexes may occur in a presence of asurfactant. An array may be formed with a display nucleic acidnanostructure that may be coupled to an analyte before or after formingan array. In some configurations, an analyte may be covalently coupledto a display moiety.

An array comprising nucleic acid nanostructures or nucleic acidnanostructure complexes may be formed under a particular temperatureconfiguration. For example, a first SNAP or SNAP complex may be combinedwith a second SNAP or SNAP complex at a first temperature, then thetemperature may be altered (e.g., decreased, increased), therebycoupling the first SNAP or SNAP complex to the second SNAP or SNAPcomplex to form an array. A step in an array formation process may occurat a temperature of at least about 0° C., 10° C., 25° C., 50° C., 75°C., 90° C., 95° C., or more than 95° C. Alternatively or additionally, astep in an array formation process may occur at a temperature of no morethan about 95° C., 90° C., 75° C., 50° C., 25° C., 10° C., 0° C., orless than 0° C. In some configurations, temperature may be utilized toincrease the specificity of nucleic acid nanostructure deposition on asurface. For example, it may be advantageous to contact a nucleic acidnanostructure comprising a plurality of surface-interactingoligonucleotides with a coupling surface comprising a plurality ofsurface-linked complementary oligonucleotides at a higher temperature,then decrease the temperature when the nucleic acid nanostructure hashad sufficient time to obtain a most-stable configuration on thecoupling surface. Surprisingly, increased temperature of nucleic acidnanostructure or nucleic acid nanostructure complex deposition mayincrease the likelihood of depositing only one nucleic acidnanostructure on a coupling surface due to the increased energyavailable for the nucleic acid nanostructure to find a position on acoupling surface where a maximal number of surface-interacting moietiescan form a binding interaction, and the increased likelihood that anoptimal deposition position for the nucleic acid nanostructure on thecoupling surface will obstruct other nucleic acid nanostructures fromco-depositing stably on the same coupling surface.

A nucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof), or an analyte-coupled version thereof,as set forth herein, may be deposited on a surface or solid support. Themethods and compositions set forth below will generally be exemplifiedwith reference to a SNAP or SNAP complex; however, it will be understoodthat the examples can be extended to any nucleic acid, as set forthherein, including a population having the same species of SNAP or SNAPcomplex, a population having different species of SNAP or SNAP complex,a population having the same species of analyte-coupled SNAP or SNAPcomplex, or a population having different species of analyte-coupledSNAP or SNAP complexes.

In an aspect, provided herein is a method comprising: a) contacting anucleic acid, as set forth herein, with a solid support, as set forthherein; and b) coupling the nucleic acid to the solid support. In somecases, a method may comprise the steps of: a) providing a solid support,as set forth herein, in which the solid support comprises a site and aninterstitial region, in which the site is configured to couple a nucleicacid, as set forth herein, and in which the interstitial region isconfigured to inhibit binding of a nucleic acid, b) contacting the solidsupport with the nucleic acid, and c) coupling the nucleic acid to thesite of the solid support. In some cases, a method may comprise thesteps of: a) providing a solid support, as set forth herein, in whichthe solid support comprises a plurality of sites and one or moreinterstitial regions, in which a site of the plurality of sites isconfigured to couple a nucleic acid, as set forth herein, and in whichthe interstitial region is configured to inhibit binding of a nucleicacid, b) contacting the solid support with a plurality of nucleic acids,in which the plurality of nucleic acids comprises the nucleic acid, andc) coupling the nucleic acid of the plurality of nucleic acids to thesite of the plurality of sites.

An analyte may be coupled to a SNAP or SNAP complex before, during, orafter deposition of the SNAP or SNAP complex on a surface or solidsupport. The deposition of a SNAP or SNAP complex on a surface or solidsupport may be driven by a physical phenomenon such as gravity,centrifugal force, electrostatic interactions, magnetic interactions,covalent binding, or non-covalent binding. In some cases, the depositionof a SNAP or SNAP complex may be due to the electrostatic interactionbetween a negatively-charged SNAP or SNAP complex and apositively-charged substrate (or other material), or vice versa. Inother cases, the deposition of a SNAP or a SNAP complex may be due tocoupling interactions between a plurality of surface-interactingmoieties on a SNAP with a plurality of surface-linked moieties on acoupling surface.

Before a SNAP, a SNAP complex, or an analyte-coupled version thereof iscoupled to a solid support, the SNAP, SNAP complex, or analyte-coupledversion thereof may be purified. In some cases, purification maycomprise removal of excess or unwanted reagents (e.g., salts, unboundoligonucleotide, unbound analytes, etc.). In some cases, a purificationprocess may comprise removal of SNAPs or SNAP complexes that do notcomprise a coupled analyte. In some cases, a purification process maycomprise removal of SNAPs or SNAP complexes that comprise more than onecoupled analyte. In some cases, a purification process may compriseremoval of analytes that are coupled to more than one SNAP or SNAPcomplex. A SNAP, a SNAP complex, or an analyte-coupled version thereofmay be purified by a suitable purification process, such as sizeexclusion chromatography, high-pressure liquid chromatography,ultrafiltration, tangential flow filtration, reverse osmosis, affinitychromatography, or combinations thereof. A plurality of analytes orSNAP-analyte composites may be characterized based upon a statistical orstochastic measure of purity. In some cases, a plurality of analytes maybe provided for preparation of an array of analytes if a measure ofpurity deviates from an expected measure of purity for a statistical orstochastic distribution (e.g., a Poisson distribution, a normaldistribution, a binomial distribution, etc.), in which the statisticalor stochastic distribution is calculated for a situation of a singleanalyte coupled to a single nucleic acid. For example, a plurality ofanalytes coupled to a plurality of nucleic acid nanostructures may beutilized for a method, as set forth herein, if a purified fractioncontains less than 36.8% nucleic acid nanostructures that are notcoupled to an analyte (e.g., a lower ratio than predicted by a Poissondistribution). A purified plurality of analytes may be characterizedwith respect to fraction of unoccupied nucleic acids, fraction ofnucleic acids with more than one analyte, fraction of analytes coupledto more than one nucleic acid, or combinations thereof.

SNAPs, SNAP complexes, or analyte-coupled versions thereof may bedeposited on a surface or solid support to form a patterned, ordered, orunordered array of SNAPs, SNAP complexes, or analyte-coupled versionsthereof. In some cases, the surface or solid support may be structured,engineered, or fabricated to control where the deposition of SNAPs orSNAP complexes may occur. The surface or solid support may containlocalized or uniform regions of positive or negative surface chargedensity that promote electrostatic interactions with a SNAP or SNAPcomplex. A surface or solid support may be deposited with a coating,layer, or functional group that alters the surface charge density of thesurface or material to promote electrostatic interactions with ananchoring group of a protein conjugate. A surface or solid support maybe functionalized with a chemical species that permits direct covalentattachment of a SNAP or SNAP complex to the surface or material.Exemplary surfaces and solid supports that can be particularly usefulare set forth elsewhere herein.

A deposition of SNAPs, SNAP complexes, or analyte-coupled versionsthereof, as set forth herein, on a surface or solid support material maybe controlled to ensure sufficient separation between neighboring SNAPsor SNAP complexes. For an analyte assay, SNAPs, SNAP complexes, oranalyte-coupled versions thereof may be deposited with sufficientseparation to ensure that each SNAP, SNAP complex, or analyte-coupledversion thereof is located at a unique, optically observable address orlocation on a surface or solid support. Separation between neighboringSNAPs, SNAP complexes, or analyte-coupled versions thereof may becontrolled by the surface or solid support material; the SNAPs, SNAPcomplexes, or analyte-coupled versions thereof, or by a combinationthereof. For example, features may be present on a surface and eachfeature may have dimensions or chemical functionalization(s) thataccommodate only a single SNAP or SNAP complex. Alternatively oradditionally, functional groups may be present on SNAPs or SNAPcomplexes in an orientation that limits the arrangement of the SNAPs orSNAP complexes on a surface that is reactive to the functional groups. Asurface or solid support material may be modified to mediate thedeposition of SNAPs, SNAP complexes, or analyte-coupled versions thereofat binding sites. Areas of the surface or solid support between bindingsites may be modified to discourage or prevent deposition of SNAPs, SNAPcomplexes, or analyte-coupled versions thereof. Deposition of SNAPs,SNAP complexes, or analyte-coupled versions thereof may be prevented bysurface groups or materials that sterically obstruct a protein conjugatefrom depositing on the surface, such as tethered dextrans, tetheredpolyethylene glycol (PEG) macromolecules or sheared salmon sperm DNA.Deposition of SNAPs, SNAP complexes, or analyte-coupled versions thereofto particular regions on a surface, such as interstitial regions whichare intended to separate addresses where SNAPs are to reside, may beprevented by surface groups that electrostatically or magnetically repelof SNAPs, SNAP complexes, or analyte-coupled versions thereof. Forexample, a negatively charged SNAP or SNAP complex may be repelled fromareas of a substrate surface that have been functionalized withnegatively charged groups such as a carboxylic acids, organophosphates,organosulfates, or combinations thereof. In some cases, solventconfiguration may be utilized to facilitate and/or inhibit SNAPdeposition at areas of a surface or solid support. For example, salts,surfactants, or emulsions may be utilized to areas of more favorable orless favorable binding conditions.

Covalent bonds may be formed between a SNAP, SNAP complex, oranalyte-coupled version thereof, as set forth herein, and a surface orsolid support. A covalent bond may be formed directly between a SNAP,SNAP complex, or analyte-coupled version thereof and a surface or solidsupport. A covalent bond may be formed between a functional group on aSNAP, SNAP complex, or analyte-coupled version thereof and a surface orsolid support. For example, a SNAP, SNAP complex, or analyte-coupledversion thereof functionalized with an organosilane group may be bondedto a silicon surface or solid support by a coordination bond. A covalentbond may be formed between a functional group on a SNAP, SNAP complex,or analyte-coupled version thereof and a functional group on a surfaceor solid support. For example, a SNAP, SNAP complex, or analyte-coupledversion thereof containing an activated ester functional group may bebonded to a surface or solid support containing an aminated functionalgroup (e.g., 3 amino-propyl triethoxysilane, silanol, etc.). In somecases, a SNAP, SNAP complex, or analyte-coupled version thereof may becoupled to a solid support or surface by a covalent bond formed by aClick-type reaction.

A SNAP or a SNAP complex, as set forth herein, may be deposited on amaterial, surface, or solid support comprising an ordered or unorderedsurface. An ordered surface may comprise a surface that is patternedwith a plurality of binding sites or regions separated by interstitialregions, where each binding site may be configured to bind a SNAPcomplex, and where the interstitial regions may be configured to notbind the SNAP complex. In some configurations, a surface or solidsupport may comprise a patterned array. An ordered surface mayfacilitate deposition of SNAPs or SNAP complexes by limiting regionswhere SNAPs or SNAP complexes may deposit, or by providing orderedfeatures that encourage the deposition of SNAPs or SNAP complexes. Inother configurations, an unordered surface may comprise a surface withno patterned or structured features. For example, a surface may comprisea uniform coating or layer of functional groups or moieties that areconfigured to couple SNAPs or SNAP complexes. In some configurations, anunordered surface may comprise a phase boundary between two fluids, suchas a gas/liquid interface or a liquid/liquid interface. In otherconfigurations, an unordered surface may comprise a mobile layer (e.g.,a lipid monolayer or bilayer, a layer of tethered or adhered micelles orcolloids, etc.). SNAPs or SNAP complexes may be configured toself-assemble or self-pattern on an unordered surface. For example,SNAPs or SNAP complexes may comprise utility moieties on one or morefaces that sterically block the approach of other SNAPs or SNAPcomplexes, thereby limiting the ability for two SNAP to co-locate withina region of steric occlusion or obstruction.

A material may comprise a surface or solid support that is patterned orstructured with binding sites or regions and interstitial regions toform a patterned array of SNAPs or SNAP complexes. In someconfigurations, individual binding sites may further comprise structuresthat facilitate the deposition of SNAPs or SNAP complexes at the bindingsite or region, and/or limit or prevent the co-deposition of multipleSNAPs or SNAP complexes at the binding site or region. Surface featuresthat may be altered to facilitate SNAP or SNAP complex deposition mayinclude binding site or region size, binding site or region morphology,and binding site or region chemistry. In some configurations, a solidsupport, a surface thereof, and/or a site thereof may comprise atwo-dimensional and/or three-dimensional feature that facilitatesbinding of a SNAP or SNAP complex to the surface. In particularconfigurations, a two-dimensional and/or three-dimensional feature maycomprise a shape or morphology that substantially matches a shape ormorphology of a SNAP and/or SNAP complex. A shape or morphology of asolid support, a surface thereof, and/or a site thereof may match ashape or morphology of a SNAP if the shape or morphology has asubstantially similar surface area to an effective surface area orfootprint of a SNAP, SNAP complex, or a face thereof. A shape ormorphology of a solid support, a surface thereof, and/or a site thereofmay match a shape or morphology of a SNAP if the shape or morphology hasa surface contour that substantially align with a contour of a SNAP,SNAP complex, or a face thereof. For example, a triangular SNAP may bedeposited on a triangular site. In another example, a site may comprisea pyramidal, three-dimensional raised structure that couples to apyramidal void space of a SNAP structure. In other particularconfigurations, a two-dimensional and/or three-dimensional feature maycomprise a shape or morphology that does not substantially match a shapeor morphology of a SNAP and/or SNAP complex.

In some configurations, a SNAP or SNAP complex may have a shape orconformation that limits the deposition of SNAPs or SNAP complexes at abinding site or region. FIG. 26A depicts a binding site 2600 comprising2 electrostatically-bound cross-shaped SNAP complexes 2610. Althoughboth SNAP complexes 2610 each occupy less than 25% of the surface areaof the binding site 2600, the cross-shaped conformation limits theability for more than two SNAP complexes to deposit with sufficientsurface contact to form a stable electrostatic binding interaction. FIG.26B depicts a binding site 2600 comprising 2 electrostatically-boundstar-shaped SNAP complexes 2620. Although the combined footprint of the2 SNAP complexes 2620 is less than the total footprint of the bindingsite 2600, the conformation of the first complex prevents the secondcomplex from fully occupying the binding site, increasing the likelihoodthat the second complex may dissociate from the binding site 2600. Thus,the first SNAP complex 2610 to occupy the binding site 2600 willsterically block a second SNAP complex 2610 from co-occupying thebinding site 2600. In some configurations, a conformation of a firstSNAP or SNAP complex coupled to a binding site or region may prevent asecond SNAP or SNAP complex from coupling to the binding site. FIG. 26Cdepicts a binding site 2600 comprising a SNAP complex 2630 comprising 21tile-shaped SNAPs that fully occupies the binding site such that noother SNAP complexes may deposit at the binding site 2600.

Binding sites or regions may also be configured to facilitate SNAP orSNAP complex deposition due to binding site or region morphology.Binding sites or regions may comprise raised pedestals, wells, ordepressions. Surface discontinuities (e.g., edges or boundaries) thatform pedestals or wells may limit the deposition of SNAPs due toenergetic effects. Without wishing to be bound by theory, SNAPs or SNAPcomplexes may be less likely to deposit adjacent to edges ordiscontinuities if portions of the SNAP or SNAP complex may beincompletely in contact with a binding surface. Reducing the size of abinding site or region may also increase the likelihood that only asingle SNAP or SNAP complex may favorably bind to a binding site orregion. Binding sites or region may further comprise small-scalefeatures that encourage SNAP or SNAP complex deposition within thebinding site or region. FIGS. 28A-28B depict raised surface features2800 that are matched to the conformation of capture faces 2820 on SNAPcomplexes 2810. Such features may be created by lithographic ordepositional techniques to from more specific features to bind SNAPs orSNAP complexes at a binding site. Multiple types of patterned surfacefeatures may be utilized to segregate different SNAP or SNAP complextypes on a surface. FIG. 27A depicts a surface 2700 comprising 6 bindingsites 2710. Two binding sites are patterned with a triangular surfacefeature 2715 and 4 binding sites are patterned with a square surfacefeature 2718. As shown in FIG. 27B, after the surface has bee contactedwith a mixture of triangular and square SNAP complexes, the triangularSNAP complexes 2725 preferentially bind to the triangular surfacefeatures 2715 and the square SNAP complexes 2728 preferentially bind tothe square surface features 2718.

The surface chemistry of a binding site or binding region may also beconfigured to facilitate SNAP or SNAP complex deposition. A binding siteor binding region may include localized regions of functional groups ormoieties that are configured to couple a SNAP or SNAP complex (e.g.,click reactive groups, oligonucleotides, etc.). A binding site orbinding region may further comprise regions of blocking or passivatinggroups that discourage the specific or non-specific binding of SNAPs orSNAP complexes to particular portions of a binding site or region (e.g.,edges, boundaries). Localized surface chemistries may be generated byany suitable technique, including deposition and lift-off techniques.Further surface chemistry methods are discussed in PCT/US2020/058416,which is hereby incorporated by reference in its entirety. In somecases, distribution or density of a two or more species of functionalgroups or moieties (e.g., surface-linked moieties) may be controlled bydeposition of mixtures of the two or more species at relativeconcentrations that produce the desired surface distribution or surfacedensity of each respective species. For example, a coupling surfacecomprising two surface-linked oligonucleotides with a 1:100 molar ratiomay be formed by co-depositing the oligonucleotides from a fluidicmedium comprising the two oligonucleotides in an approximately 1:100molar ratio. In some cases, relative ratios of species may be adjusteddue to kinetic differences in deposition.

A plurality of SNAPs, SNAP complexes, or analyte-coupled versionsthereof may be deposited on a surface or solid support with a known orcharacterized efficiency. In certain cases where the available number ofbinding sites on a surface or substrate exceeds the population size ofthe plurality of SNAPs, SNAP complexes, or analyte-coupled versionsthereof, the efficiency of deposition may be measured based upon thefraction of the plurality of SNAPs, SNAP complexes, or analyte-coupledversions thereof that are deposited on the surface or solid support. Incertain cases where the plurality of SNAPs, SNAP complexes, oranalyte-coupled versions thereof exceeds the available number of bindingsites on a surface or solid support, the efficiency of deposition may bemeasured based upon the fraction of available binding sites on thesurface or solid support that are occupied after deposition.

The binding efficiency of a plurality of SNAPs, SNAP complexes, oranalyte-coupled versions thereof to a surface or solid support may bequantified based upon a percentage or fraction of the plurality ofSNAPs, SNAP complexes, or analyte-coupled versions thereof that aredeposited on the surface or solid support. The binding efficiency of aplurality of SNAPs, SNAP complexes, or analyte-coupled versions thereofmay be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%,99.999999%, or more than 99.999999% based upon the available number ofSNAPs, SNAP complexes, or analyte-coupled versions thereof in theplurality. Alternatively or additionally, the binding efficiency of aplurality of SNAPs, SNAP complexes, or analyte-coupled versions thereofmay be no more than about 99.999999%, 99.99999%, 99.9999%, 99.999%,99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, 1%, or less than about 1% based upon the available numberof SNAPs, SNAP complexes, or analyte-coupled versions thereof in theplurality.

The binding efficiency of a plurality of SNAPs, SNAP complexes, oranalyte-coupled versions thereof to a surface or solid support may bequantified based upon a percentage or fraction of the available bindingsites on the surface or solid support that become occupied with a SNAP,SNAP complex, or analyte-coupled version thereof. The occupancy rate ofsurface or solid support binding sites may be at least about 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, 99.999999%, or more than99.999999% based upon the total number of available binding sites.Alternatively or additionally, the occupancy rate of surface or solidsupport binding sites may be no more than about 99.999999%, 99.99999%,99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%,93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than about 1% based uponthe total number of available binding sites.

In particular configurations, more than one SNAP, SNAP complex, oranalyte-coupled version thereof, as set forth herein, may deposit on asurface or solid support at a unique location, address, or binding siteon the surface or solid support. In some cases, the number of bindingsites with more than one SNAP, SNAP complex, or analyte-coupled versionthereof may be minimized to accommodate single molecule detection duringan analyte assay. In other cases, more than one SNAP, SNAP complex, oranalyte-coupled version thereof may be deposited at a plurality,majority, or at all available binding sites, such as during a bulkanalyte assay. A surface or solid support comprising a plurality ofdeposited SNAPs, SNAP complexes, or analyte-coupled versions thereof maybe characterized or quantified to determine the number of binding siteswith more than one SNAP, SNAP complex, or analyte-coupled versionthereof. A surface or solid support binding site may contain more thanone SNAP, SNAP complex, or analyte-coupled version thereof, such as, forexample, about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more SNAPs, SNAPcomplexes, or analyte-coupled versions thereof. Binding sites with morethan one deposited SNAP, SNAP complex, or analyte-coupled versionthereof may exist according to some quantifiable distribution, such as aPoisson distribution, binomial distribution, beta-binomial distribution,hypergeometric distribution, or bimodal distribution.

The percentage of binding sites on a surface or solid support with morethan one SNAP, SNAP complex, or analyte-coupled version thereof may bequantified based upon the observed number of molecules detected at eachunique location on the surface or solid support. The number of excessmolecules at a unique location on a surface or solid support may bequantified by detection of excess fluorescence, luminescence,scintillation, or size (e.g., as characterized by atomic forcemicroscopy). The percentage of binding sites on a surface or solidsupport with more than one SNAP, SNAP complex, or analyte-coupledversion thereof may be no more than about 50%, 40%, 30%, 20%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%,0.001%, 0.0001%, 0.00001%, 0.000001%, 0.0000001%, or less than about0.0000001% of all available binding sites. Alternatively oradditionally, the percentage of binding sites on a surface or solidsupport with more than one SNAP, SNAP complex, or analyte-coupledversion thereof may be at least about 0.0000001%, 0.000001%, 0.00001%,0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more than about 50% of allavailable binding sites. In some cases, there may be no observed bindingsites on a surface or solid support with more than one deposited SNAP,SNAP complex, or analyte-coupled version thereof.

A SNAP, SNAP complex, or analyte-coupled version thereof may bedeposited on a surface or solid support under conditions that encouragethe deposition of the SNAP, SNAP complex, or analyte-coupled versionthereof at a binding site on the surface or solid support. Depositionmay occur under externally applied physical phenomena, such as electricfields, magnetic fields, heating, cooling, or combinations thereof. Insome cases, SNAPs, SNAP complexes, or analyte-coupled versions thereofmay be deposited on a surface or solid support under a condition thatpromotes deposition of the SNAP, SNAP complex, or analyte-coupledversion thereof. A solvent for deposition may be varied by chemicalcomposition, ionic strength, pH, electrical conductivity, magneticpermeability, heat capacity, thermal conductivity, reactivity, density,viscosity, polarity, and combinations thereof. The chemical compositionof a solvent for deposition of SNAPs, SNAP complexes, or analyte-coupledversions thereof may be varied by solvent types and amounts, salt typesand amounts, metal types and amounts, surfactant types and amounts,constituent pH, constituent pKa, and constituent reactivity. In somecases, a solvent for the deposition of SNAPs, SNAP complexes, oranalyte-coupled versions thereof may be composed to enhance theinteractions between SNAPs, SNAP complexes, or analyte-coupled versionsthereof and a surface or solid support, for example the electrostaticbonding of a SNAP, SNAP complex, or analyte-coupled version thereof.Without wishing to be bound by theory, a deposition solvent for SNAPs,SNAP complexes, or analyte-coupled versions thereof may minimize thefree energy of deposition for the SNAPs, SNAP complexes, oranalyte-coupled versions thereof. A deposition solvent may comprise adispersing agent, such as a surfactant or detergent, that reduces orprevents aggregation of SNAPs, SNAP complexes, or analyte-coupledversions thereof before deposition. In some cases, a SNAP or SNAPcomplex storage or preparation solvent composition may be utilized as adeposition solvent. A deposition solvent may be configured to increase alikelihood of SNAP and/or SNAP complex deposition at a preferredlocation of a surface or solid support. A deposition solvent may beconfigured to decrease a likelihood of SNAP and/or SNAP complexdeposition at a non-preferred location of a surface or solid support.

A method of depositing a nucleic acid on a solid support, as set forthherein, may be facilitated by modulating strength of a bindinginteraction between the nucleic acid and the solid support. For example,a nucleic acid may be deposited on a solid support in an initialconfiguration, then re-arranged into a more-stable final configurationby disrupting one or more existing binding interactions between thenucleic acid and the solid support at a first address of the solidsupport, and by forming one or more new binding interactions between thenucleic acid and the solid support at a second address of the solidsupport. In another example, a nucleic acid that is configured to form acovalent interaction and a non-covalent interaction with a solid supportmay first be deposited on the solid support in a fluidic medium thatinhibits the covalent interaction and facilitates the non-covalentinteraction. Then the solid support and/or nucleic acid can be contactedwith a second fluidic medium that facilitates the covalent interaction.

A method of depositing a nucleic acid may comprise modulating strengthof a binding interaction between the nucleic acid and the solid supportby altering a fluidic medium in contact with the nucleic acid and/or thesolid support. A fluidic medium, as set forth herein, may be altered bychanging a fluidic parameter, in which the fluidic parameter maycomprise any conceivable parameter, such as chemical composition (e.g.,solvent type, presence and concentration of a species such as achaotrope or surfactant, etc.), polarity, density, viscosity, boilingpoint, freezing point, pH, ionic strength, osmotic pressure, and flowrate. Modulating a strength of a binding interaction may comprise one ormore steps of: a) depositing a nucleic acid, as set forth herein, on asolid support, as set forth herein, in a first fluidic medium comprisinga first fluidic parameter, as set forth herein, b) optionally incubatingthe nucleic acid and/or the solid support in the first fluidic medium,c) contacting the nucleic acid and/or the solid support with a secondfluidic medium comprising a second fluidic parameter, in which the firstfluidic parameter and the second fluidic parameter differ, d) optionallyincubating the nucleic acid and/or the solid support in the secondfluidic medium, and e) optionally, displacing the second fluidic mediumfrom the solid support and/or the nucleic acid. In some configurations,a solid support may be contacted with a second fluidic medium beforedepositing a nucleic acid in a first fluidic medium. For example, asolid support may be incubated in a second fluidic medium that activatesa surface of the solid support for forming a binding interaction, thensubsequently contacted with a first fluidic medium comprising a nucleicacid, thereby forming the binding interaction between the nucleic acidand the surface. In some configurations, displacing a second fluidicmedium from a solid support may comprise displacing the second fluidicmedium comprising a second fluidic parameter with a first fluidic mediumcomprising a first fluidic parameter. For example, a solid support maybe incubated in a second fluidic medium that activates a surface of thesolid support for forming a binding interaction, then subsequentlycontacted with a first fluidic medium comprising a nucleic acid, therebyforming the binding interaction between the nucleic acid and thesurface. In another example, a solid support comprising a depositednucleic acid may be contacted with a second fluidic medium, therebyweakening a strength of a binding interaction between the nucleic acidand the solid support, then the second fluidic medium may be displacedby a first fluidic medium, thereby strengthening the strength of thebinding interaction between the nucleic acid and the solid support. Insome configurations, a displacing a second fluidic medium from a solidsupport may comprise displacing the second fluidic medium comprising asecond fluidic parameter with a third fluidic medium comprising a thirdfluidic parameter. For example, a second fluidic medium may be displacedby a rinsing buffer that is configured to remove any unbound entities(e.g., nucleic acids, analytes, affinity agents, reagents, etc.) from asolid support or a surface thereof. In another example, a second fluidicmedium may be displaced by a medium comprising a cross-linking agentthat is configured to couple a nucleic acid to a solid support or asurface thereof.

In some configurations, a method of modulating strength of a bindinginteraction may comprise displacing a first fluidic medium by astep-wise change to a second fluidic medium. For example, a firstfluidic medium may be withdrawn from contact with a solid support, thena second fluidic medium may be contacted with the solid support. Inother configurations, a method of modulating a strength of a bindinginteraction may comprise displacing a first fluidic medium by a gradientchange to a second fluidic medium. For example, an ionic strength of asolution in contact with a solid support may be altered from a firstionic strength to a second ionic strength by flowing a fluidic mediumpast the solid support, in which the fluidic medium undergoes a linearor non-linear gradient in concentration from the first ionic strength tothe second ionic strength. In some configurations, a method ofmodulating a strength of a binding interaction may comprise altering anenvironmental property of a fluidic medium, a solid support, and/or anucleic acid, such as a temperature, shear force, electrical field, or amagnetic field. For example, a solid support or a fluidic mediumcontacted thereto may be heated to weaken a non-covalent bindinginteraction between a nucleic acid and the solid support (e.g., anucleic acid base-pair hybridization).

A method, as set forth herein, may comprise forming a multiplexed array.A multiplexed array may comprise a first plurality of analytes and asecond plurality of analytes, in which the first plurality of analytesdiffers from the second plurality of analytes in one or more respects(e.g., sample type, sample source, analyte type, etc.). In some cases, amultiplexed array of analytes may comprise a randomly-ordered arraycomprising: a) a plurality of sites, in which each sites comprises afixed address, and b) a first plurality of analytes and a secondplurality of analytes, in which each site of the plurality of sitescomprises one and only one analyte of the first plurality of analytes orthe second plurality of analytes, and in which a spatial distribution ofsites comprising an analyte of the first plurality of analytes has arandom spatial order. In some cases, a randomly-ordered array may beformed by: a) depositing a first plurality of analytes on a solidsupport, as set forth herein, and b) after depositing the firstplurality of analytes on the solid support, depositing a secondplurality of analytes on the solid support. In other cases, arandomly-ordered array may be formed by: a) combining a first pluralityof analytes with a second plurality of analytes, and b) depositing thecombined first plurality of analytes and the second plurality ofanalytes on a solid support, as set forth herein. A first plurality ofanalytes may be distinguishable from a second plurality of analytes byone or more characteristics, such as differing nucleic acidnanostructures, differing detectable labels, differing functionalnucleic acids, or combinations thereof. In other cases, a multiplexedarray may comprise an ordered array comprising: a plurality of sites, inwhich each sites comprises a fixed address, and b) a first plurality ofanalytes and a second plurality of analytes, in which each site of theplurality of sites comprises one and only one analyte of the firstplurality of analytes or the second plurality of analytes, and in whicha spatial distribution of sites comprising an analyte of the firstplurality of analytes has a non-random spatial order. For example, anarray may be prepared with a first contiguous plurality of sites and asecond contiguous plurality of sites, in which each site of the firstcontiguous plurality of sites couples to an analyte of a first pluralityof analytes, and in which each site of the second contiguous pluralityof sites couples to an analyte of a second plurality of analytes. Insome cases, an ordered array may be formed by: a) depositing a firstplurality of analytes on a solid support, as set forth herein, and b)after depositing the first plurality of analytes on the solid support,depositing a second plurality of analytes on the solid support. Forexample, a first plurality of analytes may be deposited on a firstcontiguous region of an array and a second plurality of analytes may bedeposited on a second contiguous region of an array by a printingmethod. In other cases, an ordered array may be formed by: a) combininga first plurality of analytes with a second plurality of analytes, andb) depositing the combined first plurality of analytes and the secondplurality of analytes on a solid support, as set forth herein. Forexample, a first plurality of analytes comprising a first plurality ofnucleic acid nanostructures, and a second plurality of analytescomprising a second plurality of nucleic acid nanostructures may besimultaneously deposited on an array comprising a first plurality ofsites and a second plurality of sites, in which each site of the firstplurality of sites couples a nucleic acid nanostructure of the firstplurality of nucleic acid nanostructures, in which each site of thesecond plurality of sites couples a nucleic acid nanostructure of thesecond plurality of nucleic acid nanostructures, and in which the firstplurality of sites is spatially segregated from the second plurality ofsites.

Nucleic Acid Complexes

Described herein are nucleic acid nanostructure (e.g., SNAP) complexescomprising two or more nucleic acid nanostructures, as set forth herein.A nucleic acid nanostructure complex may comprise any structure thatcomprises a first nucleic acid nanostructure coupled to a second nucleicacid nanostructure. A nucleic acid nanostructure complex may comprise afirst nucleic acid nanostructure and a second nucleic acidnanostructure, where the first nucleic acid nanostructure is a displaynucleic acid nanostructure or a utility nucleic acid nanostructure, andwhere the second nucleic acid nanostructure is independently selectedfrom the group consisting of a display nucleic acid nanostructure and autility nucleic acid nanostructure. Accordingly, nucleic acidnanostructure complex may comprise two or more nucleic acidnanostructures each with a particular function. In some configurations,a nucleic acid nanostructure complex may comprise a utility nucleic acidnanostructure comprising a capture nucleic acid nanostructure, acoupling nucleic acid nanostructure, a structural nucleic acidnanostructure, or a combination thereof. In some configurations, anucleic acid nanostructure complex may comprise a display nucleic acidnanostructure and one or more additional nucleic acid nanostructuresthat perform a function for the nucleic acid nanostructure complex, suchas: 1) positioning the display nucleic acid nanostructure with respectto a second display nucleic acid nanostructure; 2) positioning thedisplay nucleic acid nanostructure with respect to a non-display nucleicacid nanostructure; 3) altering the display of an analyte that iscoupled to the display nucleic acid nanostructure; 4) increasing thestrength of coupling of a nucleic acid nanostructure complex to asurface; 5) increasing the size of a surface occupied by a nucleic acidnanostructure complex; 6) adding additional functions to a nucleic acidnanostructure complex (e.g., steric blocking, optical reflection orabsorbance, magnetic coupling, barcoding, etc.); 7) increasing thenumber of analytes displayed on a surface; or 8) a combination thereof.A nucleic acid nanostructure complex may comprise one or more nucleicacid nanostructures comprising a capture face or capture moiety, whereinthe capture face or capture moiety comprises one or moresurface-interacting moieties that are configured to form a couplinginteraction with a coupling surface of a solid support.

A first nucleic acid nanostructure (e.g., a SNAP) and a second nucleicacid nanostructure of a nucleic acid nanostructure complex may becoupled by one or more coupling moieties. A first nucleic acidnanostructure comprising a first coupling face may be configured tocouple with a second nucleic acid nanostructure comprising a secondcoupling face, thereby forming a nucleic acid nanostructure complex. Afirst nucleic acid nanostructure may comprise a first coupling moietycomprising one or more functional groups or moieties that are configuredto couple to a second nucleic acid nanostructure via reaction with asecond coupling moiety comprising one or more complementary functionalgroups or moieties. Two or more nucleic acid nanostructures may becoupled in a nucleic acid nanostructure complex by any suitable couplinginteraction, including covalent and non-covalent interactions.

Provided herein is a nucleic acid nanostructure complex (e.g., a SNAPcomplex), comprising two or more nucleic acid nanostructures, where eachnucleic acid nanostructure of the two or more nucleic acidnanostructures may be selected independently from the group consistingof a display nucleic acid nanostructure, a utility nucleic acidnanostructure, or a combination thereof, where the display nucleic acidnanostructure may comprise a display moiety that may be configured tocouple to an analyte, where the utility nucleic acid nanostructure maycomprise a capture moiety that may be configured to couple with asurface, and where the two or more nucleic acid nanostructures may becoupled to form the nucleic acid nanostructure complex.

Also provided herein is a nucleic acid nanostructure composition (e.g.,a SNAP composition), comprising a material comprising a surface and twoor more nucleic acid nanostructures, where each nucleic acidnanostructure of the two or more nucleic acid nanostructures may beselected independently from the group consisting of a display nucleicacid nanostructure, a utility nucleic acid nanostructure, or acombination thereof, where the display nucleic acid nanostructure maycomprise a display moiety that may be configured to couple to ananalyte, where the two or more nucleic acid nanostructures may becoupled to the surface, and where a first nucleic acid nanostructure ofthe two or more nucleic acid nanostructures may be coupled to a secondnucleic acid nanostructure of the two or more nucleic acidnanostructures, thereby forming a nucleic acid nanostructure complex. Inparticular configurations, the nucleic acid nanostructure composition isan array of nucleic acid nanostructures or nucleic acid nanostructurecomplexes. The nucleic acid nanostructures or nucleic acid nanostructurecomplexes can be attached to an analyte or other target molecule ofinterest, thereby providing an array of the analytes or molecules ofinterest. Further examples of nucleic acid nanostructure compositions(e.g., SNAP compositions) and nucleic acid nanostructure complexes thatcan form sites or addresses of an array are set forth in the followingparagraphs and elsewhere herein in the context of various arraycompositions.

Also provided herein is a nucleic acid nanostructure composition (e.g.,a SNAP composition), comprising an analyte, a display nucleic acidnanostructure, and one or more utility nucleic acid nanostructures,where the display nucleic acid nanostructure may comprise a displaymoiety that may be configured to couple to an analyte, where the utilitynucleic acid nanostructure may comprise a capture moiety that may becoupled with a surface or configured to couple with a surface, where thedisplay nucleic acid nanostructure may be coupled to the analyte, andwhere the display nucleic acid nanostructure may be coupled to the oneor more nucleic acid nanostructures, thereby forming a nucleic acidnanostructure complex.

Also provided herein is a nucleic acid nanostructure composition (e.g.,a SNAP composition), comprising a material comprising a surface, ananalyte, a display nucleic acid nanostructure, and one or more utilitynucleic acid nanostructures, where the display nucleic acidnanostructure comprises a display moiety that may be configured tocouple to an analyte, where the capture nucleic acid nanostructurecomprises a capture moiety that may be configured to couple with asurface, where the display nucleic acid nanostructure may be coupled tothe analyte, where the display nucleic acid nanostructure may be coupledto the one or more nucleic acid nanostructures, thereby forming anucleic acid nanostructure complex, and where the nucleic acidnanostructure complex may be coupled to the surface.

A nucleic acid nanostructure complex (e.g., a SNAP complex), as setforth herein, may comprise a display nucleic acid nanostructure and autility nucleic acid nanostructure. The utility nucleic acidnanostructure may comprise a nucleic acid nanostructure selected fromthe group consisting of a capture nucleic acid nanostructure, a couplingnucleic acid nanostructure, a structural nucleic acid nanostructure, ora combination thereof. A nucleic acid nanostructure complex may comprisea display nucleic acid nanostructure and one or more capture nucleicacid nanostructures that are configured to couple the nucleic acidnanostructure complex to a surface. A nucleic acid nanostructure complexmay comprise a display nucleic acid nanostructure and one or morecoupling nucleic acid nanostructures that are configured to bind thenucleic acid nanostructure complex to a second nucleic acidnanostructure or a second nucleic acid nanostructure complex. A nucleicacid nanostructure complex may comprise a display nucleic acidnanostructure and one or more utility nucleic acid nanostructures.

A nucleic acid nanostructure (e.g., a SNAP complex), as set forthherein, may comprise a display nucleic acid nanostructure that iscoupled to, or configured to couple to, an analyte. A nucleic acidnanostructure complex may comprise a utility nucleic acid nanostructurethat is configured to couple to a surface. In some configurations, anucleic acid nanostructure may comprise a nucleic acid nanostructure asdescribed by any of the configurations described herein, for example aSNAP comprising a multifunctional moiety.

A nucleic acid nanostructure complex (e.g., a SNAP complex), as setforth herein, may comprise a display nucleic acid nanostructure or autility nucleic acid nanostructure that comprises a detectable label. Insome configurations, a display nucleic acid nanostructure or a utilitynucleic acid nanostructure may comprise a utility face, where theutility face comprises a capture moiety, a detectable label, or asterically blocking moiety. Any of a variety of detectable labels maycomprise a fluorescent label, a luminescent label, a nucleic acidbarcode, a nanoparticle label, an isotope, or a radiolabel.

A first nucleic acid nanostructure and a second nucleic acidnanostructure may be coupled by one or more coupling moieties. In someconfigurations, a display nucleic acid nanostructure may comprise afirst nucleic acid nanostructure coupling moiety and a utility nucleicacid nanostructure may comprise a second nucleic acid nanostructurecoupling moiety, where the display nucleic acid nanostructure may becoupled to the capture nucleic acid nanostructure by a coupling of thefirst nucleic acid nanostructure coupling moiety to the second nucleicacid nanostructure coupling moiety. In some configurations, a firstnucleic acid nanostructure coupling moiety and a second nucleic acidnanostructure coupling moiety may form a covalent bond, for example,between a complementary pair of click-type reaction moieties. In otherconfigurations, a first nucleic acid nanostructure coupling moiety and asecond nucleic acid nanostructure coupling moiety can form anon-covalent bond, such as a hydrogen bond, a nucleic acid base pairbond, or a streptavidin-biotin bond.

A nucleic acid nanostructure complex (e.g., a SNAP complex), as setforth herein, may comprise two or more types of nucleic acidnanostructures in specific quantities. In some configurations, a nucleicacid nanostructure complex comprises a plurality of utility nucleic acidnanostructures and a single display nucleic acid nanostructure. In somecases, a nucleic acid nanostructure complex may comprise a particularnumber of a type of nucleic acid nanostructure (e.g., a display SNAP, autility SNAP). A nucleic acid nanostructure complex may comprise atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, or more than 100 of a particular number ofa type of nucleic acid nanostructure. Alternatively or additionally, anucleic acid nanostructure complex may comprise no more than about 100,95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or less than 2 of a particular number of a type of nucleic acidnanostructure.

In some cases, a nucleic acid nanostructure complex (e.g., a SNAPcomplex) may comprise a first type of nucleic acid nanostructure (e.g.,a display SNAP) and a second type of SNAP (e.g., a utility SNAP) in afixed ratio. A nucleic acid nanostructure complex may comprise a firsttype of nucleic acid nanostructure and a second type of nucleic acidnanostructure in a ratio of at least about 1:1, 1.1:1, 1.25:1, 1.5:1,1.75:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1,25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1,37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1,49:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1,or more than 100:1. Alternatively or additionally, a nucleic acidnanostructure complex may comprise a first type of nucleic acidnanostructure and a second type of nucleic acid nanostructure in a ratioof at most about 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1,55:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1,39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1,27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1,15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,2.5:1, 2:1, 1.75:1, 1.5:1, 1.25:1, 1.1:1, or less than 1.1:1.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may comprisea first type of nucleic acid nanostructure (e.g., a display SNAP) and asecond type of nucleic acid nanostructure (e.g., a utility SNAP), wherethe second type of nucleic acid nanostructure is coupled to a particularface of the first type of nucleic acid nanostructure (e.g., a couplingface). In some configurations, a nucleic acid nanostructure complex maycomprise a first type of nucleic acid nanostructure and two or more of asecond type of nucleic acid nanostructure coupled to one or more facesof the first type of nucleic acid nanostructure. In some configurations,a nucleic acid nanostructure complex may comprise a display nucleic acidnanostructure and two or more of a utility nucleic acid nanostructurecoupled to one or more faces of the display nucleic acid nanostructure.In some configurations, a first utility nucleic acid nanostructure ofthe two or more utility nucleic acid nanostructures may be coupled to afirst face of the display nucleic acid nanostructure, and a secondutility nucleic acid nanostructure of the two or more utility nucleicacid nanostructures may be coupled to a second face of the displaynucleic acid nanostructure. In some configurations, a face of the firstutility nucleic acid nanostructure is coupled to a face of the secondutility nucleic acid nanostructure. In some configurations, a firstutility nucleic acid nanostructure is not coupled to a second utilitynucleic acid nanostructure. In some configurations, a nucleic acidnanostructure complex further comprises a third utility nucleic acidnanostructure. In some configurations, a third utility nucleic acidnanostructure is coupled to a third face of the display nucleic acidnanostructure. In some configurations, a third utility nucleic acidnanostructure is coupled to a face of a first utility nucleic acidnanostructure, a face of the second utility nucleic acid nanostructure,or a combination thereof.

A nucleic acid nanostructure complex (e.g., a SNAP complex), as setforth herein, may comprise two or more nucleic acid nanostructures withdiffering sizes or shapes, as determined on the basis of a minimum,average, or maximum measure, where the measure is, for example, length,width, depth, circumference, diameter, effective surface area,footprint, effective occupied volume, any measure of structuremorphology, or a combination thereof. A nucleic acid nanostructurecomplex may comprise a first nucleic acid nanostructure (e.g., a displaySNAP, or utility SNAP) comprising a first coupling face that is coupledto a second nucleic acid nanostructure (e.g., a display SNAP, or utilitySNAP) comprising a second coupling face, where the first coupling faceand the second coupling face have differing sizes, dimensions, ormorphologies. In various configurations, a coupling face of a firstnucleic acid nanostructure is smaller than, larger than, or the samesize as a coupling face of a second nucleic acid nanostructure. Anucleic acid nanostructure complex may further comprise a third nucleicacid nanostructure (e.g., a display SNAP, a utility SNAP) comprising athird coupling face that is coupled to the first nucleic acidnanostructure. In some configurations, a coupling face of a thirdnucleic acid nanostructure is smaller than, larger than or the same sizeas a coupling face of a first nucleic acid nanostructure.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may comprisea first nucleic acid nanostructure (e.g., a display SNAP, or utilitySNAP) comprising a first coupling face and a second nucleic acidnanostructure (e.g., a display SNAP, or utility SNAP) comprising asecond coupling face, where the first nucleic acid nanostructure and/orthe second nucleic acid nanostructure comprise a display moiety and/or acapture moiety. In some configurations, a first coupling face and asecond coupling face do not comprise a capture moiety. In someconfigurations, a first coupling face and a second coupling face do notcomprise a display moiety. In some configurations, a capture moiety maycomprise a plurality of surface-interacting moieties.

A nucleic acid nanostructure in a nucleic acid nanostructure complex(e.g., a SNAP complex) may comprise one or more coupling faces that areconfigured to couple the nucleic acid nanostructure to a second nucleicacid nanostructure. A nucleic acid nanostructure in a nucleic acidnanostructure complex may comprise at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20coupling faces. Alternatively or additionally, a SNAP in a SNAP complexmay comprise no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 coupling faces. In someconfigurations, each coupling face of a nucleic acid nanostructure in anucleic acid nanostructure complex may be coupled to a second nucleicacid nanostructure. In some configurations, at least one coupling faceof a nucleic acid nanostructure in a nucleic acid nanostructure complexis coupled to a second nucleic acid nanostructure. In someconfigurations, at least one coupling face of a nucleic acidnanostructure in a nucleic acid nanostructure complex is not coupled toa second nucleic acid nanostructure.

A nucleic acid nanostructure complex (e.g., a SNAP complex) containingtwo or more nucleic acid nanostructures, as set forth herein, may beconfigured to comprise a particular symmetry, such as a mirror symmetryor a rotational symmetry. A symmetry of a nucleic acid nanostructurecomplex may be determined with respect to average dimensions, shapes, orconfigurations of nucleic acid nanostructures within a nucleic acidnanostructure complex. Variations in positioning of features, forexample, due to the helical structure and tertiary structures of a SNAP,may result in small differences between two opposed features of a SNAPcomplex that is designed to have a symmetrical structure. A symmetricnucleic acid nanostructure may have two symmetric features which liewithin about 10% of the expected position with respect to an axis orplane of symmetry.

Symmetry may facilitate one or more functions of nucleic acidnanostructures or nucleic acid nanostructure complexes (e.g., SNAPcomplexes). Symmetry can be characterized with respect to referenceplanes that are imaginary constructs for purposes of demonstration. Insome aspects, a nucleic acid nanostructure (e.g., a SNAP) may beconfigured to have symmetry with respect to certain reference planes oraxes of rotation and this symmetry can optionally facilitate increasedflexibility or molecular motion. A nucleic acid nanostructure complexmay be further configured with one or more planes of alignment. A planeof alignment may comprise a reference plane to which one or morecoupling faces are aligned. A plane of alignment may encompass acontinuous surface in which a first nucleic acid nanostructure has somedegree of bending, flexing, or deformation with respect to a secondnucleic acid nanostructure. A nucleic acid nanostructure may be designedwith symmetry to permit assembly into particular shapes or conformationsof nucleic acid nanostructure complexes. A nucleic acid nanostructurecomplex may possess a particular symmetry that facilitates coupling to asite on a surface that is configured to couple with the complex.

A nucleic acid nanostructure or nucleic acid nanostructure complex, asset forth herein, may be asymmetric generally or with respect to certainreference planes or axes of rotation. For example, a SNAP or SNAPcomplex may possess asymmetry in a particular orientation, or maypossess no planes or axes of symmetry. An asymmetric nucleic acidnanostructure or nucleic acid nanostructure complex may provide theadvantage of being more rigid than a symmetric nucleic acidnanostructure or nucleic acid nanostructure complex, for example, due todecreased range of motion for individual nucleic acid nanostructures inthe asymmetric complex. Asymmetry in a nucleic acid nanostructure ornucleic acid nanostructure complex may also facilitate the function ofthe nucleic acid nanostructure or nucleic acid nanostructure complex.For example, asymmetry in the structure of top and bottom SNAP faces mayfacilitate differential coupling of bottom faces to a surface and topfaces to display a SNAP.

FIGS. 12A-12C illustrate aspects of SNAP and SNAP complex configurationrelating to symmetry. FIG. 12A shows a SNAP complex formed from acoupling of four utility SNAPs 1210 to a central display SNAP 1220. Eachutility SNAP 1210 is coupled to the display SNAP 1220 by coupling of acoupling face on the utility SNAP 1210 to a coupling face on the displaySNAP 1220. The coupling faces for both the utility SNAPs 1210 and thedisplay SNAPs 1220 have an effective surface area of about the multipleof the average side length and the average SNAP thickness. The SNAPcomplex formed by the coupling of the four utility SNAPs 1210 to thedisplay SNAP 1220 has two planes of symmetry indicated by referenceplanes 1230. FIG. 12B shows a cross-sectional view of the firstconfiguration of the SNAP complex. The utility SNAPs 1210 are coupled tothe display SNAP 1220 with sufficient rigidity to create a nearlycoplanar alignment between bottom faces of the SNAPs in the SNAPcomplex. The SNAP complex retains a left-right symmetry around referenceplane 1230 but lacks a top-bottom symmetry due to differences inconfiguration. The SNAP complex also comprises planes of alignmentindicated by reference planes 1235 at the coupling faces between theutility SNAPs 1210 and display SNAP 1220. Arrows at the sides of thecross-section depict potential directions of bending or flexing of theutility SNAPs 1210 with respect to the display SNAP 1220. The utilitySNAPs 1210 and the display SNAP 1220 may comprise bottom capture facescomprising a plurality of single-stranded nucleic acids 1240 that areconfigured to facilitate coupling of the SNAP complex to a surface. Theutility SNAPs 1210 may further comprise top utility faces comprising aplurality of sterically-blocking groups 1250 that are configured toprevent adhesion of other molecules to the SNAP complex other than theanalyte 1260 that is coupled to the display SNAP 1220. FIG. 12C depictsan alternative configuration of the SNAP complex with utility SNAPs 1210coupled to the display SNAP 1220 at an angle such that the capture facesof the utility SNAPs 1210 and the display SNAP 1220 are not coplanar. Insome configurations, the coupling of SNAPs in a SNAP complex may besufficiently rigid to minimize bending or deformation at interfacesbetween SNAPs. In other cases, the coupling of SNAPs in a SNAP complexmay be sufficiently flexible to permit a SNAP to adopt multipleformations, such as shifting between the formation of FIGS. 12B and 12C.

FIGS. 13A-13D show additional aspects of symmetry and asymmetry inrelation to the formation of nucleic acid nanostructure complexes. Inparticular, configurations shown in FIGS. 13A-13D compriseconfigurations with utility SNAPs that couple to other utility SNAPs inthe SNAP complex, thereby decreasing the ability of SNAPs to bend ordeform along particular reference planes within the SNAP complexincluding, for example, reference planes positioned between coupledSNAPs. FIG. 13A depicts a substantially rectangular SNAP complex with anasymmetric configuration. The SNAP complex comprises a central displaySNAP 1310 that comprises a display moiety 1320. The SNAP complex furthercomprises four utility SNAPs (1331, 1332, 1333, 1334). Utility SNAPs1331, 1332, and 1333 are each coupled via coupling faces tocomplementary coupling faces of the display SNAP 1310. The fourthutility SNAP 1334 is not coupled directly to the display SNAP but iscoupled to the first utility SNAP 1331 and the third utility SNAP 1333.Due to the differing average dimensions of each SNAP in the complex,utility SNAPs 1331, 1332, and 1333 comprise coupling faces withdiffering dimensions. Utility SNAP 1334 comprises two separate couplingfaces that comprise the larger face on the side that couples to utilitySNAP 1331 and 1333, thereby forming a plane of alignment that isorthogonal to depicted line 1340. FIG. 13B depicts a substantiallysquare SNAP complex with an asymmetric configuration. The SNAP complexcomprises a central display SNAP 1310 that comprises a display moiety1320. The SNAP complex further comprises eight utility SNAPs, including3 small utility SNAPs 1351, 2 medium utility SNAPs 1352, and 3 largeutility SNAPs 1353. The spiral arrangement of the utility SNAPs and theincreasing size of utility SNAPs as the spiral distance increases fromthe display SNAP 1310. Each utility SNAP in the configuration is coupledto at least 3 other utility SNAPs by at least 2 coupling faces ondifferent sides of the SNAP. The configuration of FIG. 13B lacks anycoupling faces between SNAPs that span the full length of the SNAPcomplex. This configuration beneficially maintains coplanarity of theSNAPS (relative to the plane of the page for the orientation shown inFIG. 13B), because the SNAP complex comprises no uninterrupted planes ofalignment along which two adjacent SNAPs can bend or flex relative toeach other so as to deviate from coplanarity. Any bending of flexing ofa SNAP within the complex would be resisted due to the complex patternof couplings in the SNAP complex.

FIG. 13C-13D depict SNAP configurations with rotational symmetry aboutan axis that is oriented orthogonal to the display moiety 1320 of thecentral display SNAP 1310. FIG. 13C illustrates a substantially squareSNAP complex comprising a display SNAP 1310 and 8 utility SNAPs,including 4 utility SNAPs 1360 that are coupled to coupling faces of thedisplay SNAP 1310, and 4 utility SNAPs 1365 that are coupled only to thedisplay-coupled utility SNAPs 1360. FIG. 13D shows a substantiallysquare SNAP complex comprising a central display SNAP 1310 and 4triangular utility SNAPs 1370. The display SNAP is coupled to each ofthe 4 utility SNAPs 1370, and each utility SNAP 1370 is coupled to twoother utility SNAPs 1370 in addition to the display SNAP 1310. Theconfigurations depicted in FIGS. 13C-13D have a rotational symmetry suchthat a 90° rotation about the display moiety produces the sameconfiguration. However, the configurations lack any uninterrupted planesof alignment between SNAPs, thereby increasing the resistance to bendingor deformation of the SNAP complex structure (relative to the plane ofthe page for the orientation shown in FIGS. 13C-13D). Such rigidity maybe useful for increasing the stability of larger arrays comprisingmultiple coupled nucleic acid nanostructure complexes. Maintainingplanarity of a nucleic acid nanostructure capture face can beparticularly advantageous for facilitating attachment of nucleic acidnanostructure complexes to a planar surface via the capture face and formaintaining the nucleic acid nanostructure complexes in a focal planefor subsequent optical detection. Substantially rigid structures mayalso have increased binding specificity and strength when contacted witha surface comprising complementary morphologies for the nucleic acidnanostructure complex capture faces. FIGS. 14A-14B depict a SNAP complexstructure in three dimensions to demonstrate another example ofsymmetry. FIG. 14A depicts a SNAP complex comprising a central displaySNAP 1420 coupled to four rectangular utility SNAPs 1410 comprising atop coupling face and a bottom coupling face. The SNAP comprises arotational axis of symmetry through the center of the display SNAP 1420but the overlapping of the rectangular SNAPs can resist bending ordeformation of the SNAPs complex. FIG. 14B depicts a similar SNAPcomplex comprising four display SNAPs 1430 with a rotational axis ofsymmetry and overlapped top and bottom coupling faces on each displaySNAP 1430.

A nucleic acid nanostructure complex (e.g., a SNAP complex), as setforth herein, may comprise at least one axis of symmetry or one plane ofsymmetry. A nucleic acid nanostructure complex may further comprise atleast one uninterrupted plane of alignment. For example, theuninterrupted plane can be located between adjacent SNAPs and theuninterrupted plane can span the length of the SNAP complex. In someconfigurations, an axis of symmetry may comprise a rotational axis ofsymmetry or a reflection axis or plane of symmetry. In someconfigurations, a nucleic acid nanostructure complex may comprise arotational axis of symmetry and a reflection axis or plane of symmetry.In other configurations, a nucleic acid nanostructure complex maycomprise no axis or plane of symmetry. In some configurations, a nucleicacid nanostructure complex may comprise no uninterrupted planes ofalignment. Again, the uninterrupted plane can be located betweenadjacent nucleic acid nanostructures and the uninterrupted plane canspan the length of the nucleic acid nanostructure complex.

An orientation of a first nucleic acid nanostructure relative to asecond nucleic acid nanostructure in a nucleic acid nanostructurecomplex (e.g., a SNAP complex) may be controlled. In someconfigurations, a first nucleic acid nanostructure may be orientedrelative to a second nucleic acid nanostructure in a nucleic acidnanostructure complex such that a face (e.g., a capture face, a displayface, a utility face) of the first nucleic acid nanostructure issubstantially parallel or coplanar with a face (e.g., a capture face, adisplay face, a utility face) of the second nucleic acid nanostructure.In other configurations, a first nucleic acid nanostructure may beoriented relative to a second nucleic acid nanostructure in a nucleicacid nanostructure complex such that a face of the first nucleic acidnanostructure is not parallel or not coplanar with a face of the secondnucleic acid nanostructure. The orientation between two nucleic acidnanostructures may be controlled, in part, by the ability to locatecoupling moieties at specific nucleotides that comprise one or moretertiary structures of a nucleic acid nanostructure. FIG. 15A-15B depictorientation control utilizing the helical structure of DNA-based SNAPs.FIG. 15A illustrates a cross-sectional view of a first SNAP 1510 that isconfigured to be coupled to 2 second SNAPs 1520. The first SNAP 1510comprises a plurality of helical tertiary structures comprising a firstcoupling group 1530 and a second coupling group 1535. The relativeplacement of the first coupling group 1530 on the helix orients thefirst coupling group 1530 nearly orthogonal to a first coupling face1540. The relative placement of the second coupling group 1535 on thehelix orients the second coupling group 1535 at a non-orthogonal anglerelative to a second coupling face 1540. The second SNAPs 1520 comprisea plurality of helical tertiary structures comprising a complementarycoupling group 1550. FIG. 15B illustrates the conformation of a SNAPcomplex formed by coupling of the 2 second SNAPs 1520 to the first SNAP1510. Due to the relative orientation of the first coupling group 1530and the second coupling group 1535, a bottom face 1560 of one of thesecond SNAPs 1520 is coplanar with a bottom face 1562 of the first SNAP,while a bottom face 1565 of the other second SNAP 1520 is not coplanarwith bottom faces 1560 or 1562.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may comprisea particular shape based upon a two-dimensional projection onto asurface, such as a square, rectangle, triangle, circle, cross, polygon,or an irregular shape. A nucleic acid nanostructure complex may bedescribed in terms of a three-dimensional structure. A nucleic acidnanostructure complex may comprise a first nucleic acid nanostructurecomprising a first conformation (e.g., substantially square faces) and asecond nucleic acid nanostructure comprising a second conformation(e.g., substantially triangular faces, substantially rectangular faces,etc.). A nucleic acid nanostructure complex may comprise a first nucleicacid nanostructure and a second nucleic acid nanostructure where bothnucleic acid nanostructures comprise substantially similar conformations(e.g., substantially square faces, substantially triangular faces,substantially rectangular faces, etc.). A nucleic acid nanostructurecomplex may comprise one or more conformations of nucleic acidnanostructures. A nucleic acid nanostructure complex may comprise atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 conformationsof nucleic acid nanostructures. Alternatively or additionally, a nucleicacid nanostructure complex may comprise no more than about 10, 9, 8, 7,6, 5, 4, 3, 2, or less than 2 conformations of nucleic acidnanostructures.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may becoupled to, or configured to couple to, one or more analytes. A nucleicacid nanostructure complex may comprise one or more display moietiesthat are coupled to, or configured to couple to, one or more analytes. Anucleic acid nanostructure complex may comprise one or more displaynucleic acid nanostructures that are coupled to, or configured to coupleto, one or more analytes. A nucleic acid nanostructure complex may becoupled to a number of analyte molecules that is less than the number ofdisplay moieties in the nucleic acid nanostructure complex. For example,a nucleic acid nanostructure complex may only be coupled to a singleanalyte, or may be coupled to no analytes. In some configurations, adisplay moiety may be coupled to two or more analytes. In someconfigurations, two or more display moieties may be coupled to ananalyte.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may beconfigured to occupy a particular amount of surface area on a surface. Asurface area occupied by a nucleic acid nanostructure complex may bemeasured as the effective surface area or footprint created by atwo-dimensional projection of the nucleic acid nanostructure complexonto a surface. In some configurations, the effective surface area orfootprint may further include surface area of a surface or interfacethat is excluded from associating with other molecules (nucleic acidnanostructure or non-nucleic acid molecules) due to effects such assteric exclusion or repulsion caused by the nucleic acid nanostructurecomplex. A nucleic acid nanostructure complex may have an effectivesurface area or footprint of at least about 25 nm², 100 nm², 500 nm²,1000 nm², 2000 nm², 3000 nm², 4000 nm², 5000 nm², 5500 nm², 6000 nm²,6500 nm², 7000 nm², 7500 nm², 8000 nm², 8500 nm², 9000 nm², 10000 nm²,15000 nm², 20000 nm², 25000 nm², 50000 nm², 100000 nm², 250000 nm²,500000 nm², or more than 1000000 nm². Alternatively or additionally, anucleic acid nanostructure complex may have an effective surface area orfootprint of no more than about 1000000 nm², 500000 nm², 250000 nm²,100000 nm², 50000 nm², 25000 nm², 20000 nm², 15000 nm², 10000 nm², 9000nm², 8500 nm², 8000 nm², 7500 nm², 7000 nm², 6500 nm², 6000 nm², 5500nm², 5000 nm², 4000 nm², 3000 nm², 2000 nm², 1000 nm², 500 nm², 100 nm²,25 nm², or less than 25 nm².

Nucleic acid nanostructure complexes (e.g., SNAP complexes) may comprisethree-dimensional structures that improve the display of analytes.Analyte display may be improved by increasing the likelihood ofdetection and observation of an analyte, increasing the contact ofanalytes with probes or reagents, and/or decreasing negativeinteractions between analytes and other molecules. FIGS. 16A-16B depictcross-sectional views of various three-dimensional nucleic acidnanostructure complexes. FIG. 16A depicts a three-dimensional SNAPcomplex that forms a well-like structure around a central analyte. Awell-like structure may be advantageous for affinity-based assay wherethe reduction in available volume around the analyte may decrease theability of an affinity reagent to move away from the analyte.Additionally, surrounding utility SNAPs may comprise optical materialsthat increase the collection of light or decrease background signal,thereby improving the efficiency of optical detection methods. FIG. 16Bdepicts a three-dimensional SNAP complex that forms a post that elevatesan analyte above a surface to which the SNAP complex is associated. Anelevated analyte may be less likely to have unwanted interactions, forexample with molecules that may non-specifically bind to the nucleicacid nanostructure complex. An elevated analyte may also be moreaccessible to a receptor that would otherwise experience sterichindrance, charge repulsion or other inhibitory interactions with thesurface to which the nucleic acid nanostructure is attached.

Provided herein is a method of forming a nucleic acid nanostructurecomplex (e.g., a SNAP complex), comprising providing a display nucleicacid nanostructure and one or more capture nucleic acid nanostructuresor utility nucleic acid nanostructures, where the display nucleic acidnanostructure comprises one or more coupling moieties, and where thecapture nucleic acid nanostructures or utility nucleic acidnanostructures comprise one or more complementary coupling moieties,where the one or more complementary coupling moieties are configured tobe coupled with the one or more coupling moieties, and coupling thedisplay nucleic acid nanostructure to the one or more capture nucleicacid nanostructures or utility nucleic acid nanostructures by thecoupling of the one or more coupling moieties to the one or morecomplementary coupling moieties, thereby forming a nucleic acidnanostructure complex, where the nucleic acid nanostructure complexcomprises a display moiety that is configured to couple to an analyte,and where the nucleic acid nanostructure complex comprises a capturemoiety that is configured to associate with a surface. A nucleic acidnanostructure complex may comprise a display nucleic acid nanostructureand/or a utility nucleic acid nanostructure comprising a capture moietycomprising a plurality of surface-interacting moieties.

A nucleic acid nanostructure complex (e.g., a SNAP complex) formationmethod may comprise the coupling of one or more coupling moieties to oneor more complementary coupling moieties by forming a covalent bond. Insome configurations, the covalent bond is formed by performing aclick-type reaction. However, other coupling reactions and moieties canbe used such as those set forth elsewhere herein. For example, a nucleicacid nanostructure complex formation method may comprise the coupling ofthe one or more coupling moieties to the one or more complementarycoupling moieties by forming a non-covalent bond. In someconfigurations, forming a non-covalent bond comprises forming a nucleicacid base-pair hybridization. In some configurations, the one or morecomplementary coupling moieties comprise one or more oligonucleotideswith complementary sequences to the set of one or more oligonucleotides.In some configurations, forming a non-covalent bond comprises forming areceptor-ligand complex such as a streptavidin-biotin complex.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may beformed in a particular formation condition. A nucleic acid nanostructurecomplex may be formed in a fluidic medium. A condition may include aparticular solvent, polarity, ionic strength or pH buffering condition.In some configurations, a display nucleic acid nanostructure or autility nucleic acid nanostructure may be provided in a solutioncomprising a magnesium salt. In some configurations, coupling a displaynucleic acid nanostructure to one or more utility nucleic acidnanostructures may occur in the presence of a surfactant. A nucleic acidnanostructure complex may be formed with a display nucleic acidnanostructure. A display nucleic acid nanostructure may be coupled to ananalyte before or after forming a nucleic acid nanostructure complex. Insome configurations, an analyte may be covalently coupled to a displaymoiety.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may beformed under a particular temperature profile. For example, a firstnucleic acid nanostructure may be combined with a second nucleic acidnanostructure at a first temperature, then the temperature may bealtered (e.g., decreased, increased), thereby coupling the first nucleicacid nanostructure to the second nucleic acid nanostructure to form anucleic acid nanostructure complex. A step in a nucleic acidnanostructure complex formation process may occur at a temperature of atleast about 0° C., 5° C., 10° C., 15° C., 20° C., 21° C., 22° C., 23°C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32°C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77°C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86°C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95°C., or more than 95° C. Alternatively or additionally, a step in anucleic acid nanostructure complex formation process may occur at atemperature of no more than about 95° C., 94° C., 93° C., 92° C., 91°C., 90° C., 89° C., 88° C., 87° C., 86° C., 85° C., 84° C., 83° C., 82°C., 81° C., 80° C., 79° C., 78° C., 77° C., 76° C., 75° C., 74° C., 73°C., 72° C., 71° C., 70° C., 69° C., 68° C., 67° C., 66° C., 65° C., 64°C., 63° C., 62° C., 61° C., 60° C., 59° C., 58° C., 57° C., 56° C., 55°C., 54° C., 53° C., 52° C., 51° C., 50° C., 49° C., 48° C., 47° C., 46°C., 45° C., 44° C., 43° C., 42° C., 41° C., 40° C., 39° C., 38° C., 37°C., 36° C., 35° C., 34° C., 33° C., 32° C., 31° C., 30° C., 29° C., 28°C., 27° C., 26° C., 25° C., 24° C., 23° C., 22° C., 21° C., 20° C., 15°C., 10° C., 5° C., 0° C., 0° C., or less than 0° C.

A nucleic acid nanostructure complex (e.g., a SNAP complex) may compriseportions that are fully structured and/or portions that are partiallystructured. A fully structured portion of a nucleic acid nanostructurecomplex may be defined as a region of a nucleic acid nanostructurecomplex that maintains each of primary, secondary, and tertiarystructure during the course of use. A partially-structured portion of anucleic acid nanostructure complex may be defined as a region of anucleic acid nanostructure complex that comprises a primary structurebut does not maintain a particular secondary and/or tertiary structureduring the course of use. An example of a useful partially-structuredportion is a pervious structure or region of a nucleic acidnanostructure. In some configurations, a partially-structured portion ofa nucleic acid nanostructure complex may comprise a single-strandednucleic acid. A single-stranded nucleic acid may be located betweenregions of double-stranded nucleic acid, or may comprise a pendant orterminal strand of nucleic acid. A single-stranded nucleic acid maycomprise a sequence, composition or length exemplified herein forpendant nucleic acids or pendant moieties. In some configurations, apartially-structured portion of a nucleic acid nanostructure complex maycomprise an amorphous structure, such as a globular structure (e.g., ananoball, a dendrimer, etc.). FIG. 37B depicts a SNAP complex comprisinga DNA origami SNAP 3710 that is coupled to two DNA nanoball SNAPs 3735and an analyte 3720. The DNA nanoballs 3735 may be consideredpartially-structured due to their single-stranded, globular, and/oramorphous structure. Partially-structured regions of the SNAP complexmay provide one or more functionalities to the SNAP 3710 such as, forexample, increasing binding strength to targeted binding surfaces,decreasing binding strength to non-targeted surfaces, and preventnon-specific binding of other molecules to a SNAP face or a coupledanalyte.

Nucleic Acid Compositions

Nucleic acids, such as nucleic acid nanostructures, SNAPs, nucleic acidnanostructure complexes, and/or components thereof (e.g., scaffolds,staples, multifunctional moieties, etc.), as set forth herein, may bestored, prepared, or utilized in a suitable solvent or buffer. Thesolvent or buffer may provide favorable conditions for promoting thestability of nucleic acids. The solvent or buffer may facilitate aprocess, such as contacting a nucleic acid (e.g., a nucleic acidnanostructure, SNAP, a complex thereof, or a component thereof) with asurface, or contacting a nucleic acid (e.g., a nucleic acidnanostructure, SNAP, a complex thereof, or a component thereof) with ananalyte. In some configurations, a suitable DNA buffer may comprise amagnesium salt and/or EDTA. A nucleic acid may be disposed in a solventor buffer that is configured to facilitate a wanted interaction (e.g.,binding of a nucleic acid to a site of an array, etc.). A nucleic acidmay be disposed in a solvent or buffer that is configured to inhibit anunwanted interaction (e.g., aggregation of a first nucleic acid with asecond nucleic acid, etc.). An interaction of a nucleic acid (e.g.,binding to a solid support, remaining in solution, etc.) may befacilitate by a presence of a chemical species, as set forth herein. Forexample, binding of a nucleic acid to a solid support surface may bemediated by a cationic species. In another example, a surfactant speciesmay be included in a nucleic acid composition to prevent unwantedaggregation of nucleic acids, for example due to adhesion of a firstnucleic acid to an analyte that is coupled to a second nucleic acid. Amethod, as set forth herein, may utilize a fluidic medium comprising oneor more chemical species, as set forth herein. A method, as set forthherein, may comprise a step of altering a fluid medium, as set forthherein, for example by introducing or removing one or more chemicalspecies from the fluidic medium. A method, as set forth herein, maycomprise a step of exchanging a first fluidic medium, as set forthherein, for a second fluidic medium.

A solvent or buffer that is contacted with a nucleic acid (e.g., anucleic acid nanostructure, SNAP, a complex thereof, or a componentthereof) may comprise any of a variety of components, such as a solventspecies, pH buffering species, a cationic species, an anionic species, asurfactant species, a denaturing species, or a combination thereof. Asolvent species may include water, acetic acid, methanol, ethanol,n-propanol, isopropyl alcohol, n-butanol, formic acid, ammonia,propylene carbonate, nitromethane, dimethyl sulfoxide, acetonitrile,dimethylformamide, acetone, ethyl acetate, tetrahydrofuran,dichloromethane, chloroform, carbon tetrachloride, dimethyl ether,diethyl ether, 1-4, dioxane, toluene, benzene, cyclohexane, hexane,cyclopentane, pentane, or combinations thereof. A solvent or solutionmay include a buffering species including, but not limited to, MES,Tris, Bis-tris, Bis-tris propane, ADA, ACES, PIPES, MOPSO, MOPS, BES,TES, HEPES, HEPBS, HEPPSO, DIPSO, MOBS, TAPSO, TAPS, TABS, POPSO, TEA,EPPS, Tricine, Gly-Gly, Bicine, AMPD, AMPSO, AMP, CHES, CAPSO, CAPS, andCABS. A solvent or solution may include cationic species such as Na⁺,K⁺, Ag⁺, Cu⁺, NH₄ ⁺, Mg₂₊, Zn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cr²⁺, Mn²⁺, Ge²⁺,Sn²⁺, Al³⁺, Cr³⁺, Fe³⁺, Co³⁺, Ni³⁺, Ti³⁺, Mn³⁺, Si⁴⁺, V⁴⁺, Ti⁴⁺, Mn⁴⁺,Ge⁴⁺, Se⁴⁺, V⁵⁺, Mn⁵⁺, Mn⁶⁺, Se⁶⁺, and combinations thereof. A solventor solution may include anionic species such as F⁻, Cl⁻, Br⁻, ClO₃ ⁻,H₂PO₄ ⁻, HCO₃ ⁻, HSO₄ ⁻, OH⁻, I⁻, NO₃ ⁻, NO₂ ⁻, MnO₄ ⁻, SCN⁻, CO₃ ²⁻,CrO₄ ²⁻, Cr₂O₇ ²⁻, HPO₄ ²⁻, SO₄ ²⁻, SO₃ ²⁻, PO₄ ³⁻, and combinationsthereof. A solvent or solution may include a surfactant speciesincluding, but not limited to, stearic acid, lauric acid, oleic acid,sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, dodecylaminehydrochloride, hexadecyltrimethylammonium bromide, polyethylene oxide,nonylphenyl ethoxylates, Triton X, pentapropylene glycol monododecylether, octapropylene glycol monododecyl ether, pentaethylene glycolmonododecyl ether, octaethylene glycol monododecyl ether, lauramidemonoethylamine, lauramide diethylamine, octyl glucoside, decylglucoside, lauryl glucoside, Tween 20, Tween 80,n-dodecyl-β-D-maltoside, nonoxynol 9, glycerol monolaurate,polyethoxylated tallow amine, poloxamer, digitonin, zonyl FSO,2,5-dimethyl-3-hexyne-2,5-diol, Igepal CA630, Aerosol-OT, triethylaminehydrochloride, cetrimonium bromide, benzethonium chloride, octenidinedihydrochloride, cetylpyridinium chloride, adogen,dimethyldioctadecylammonium chloride, CHAPS, CHAPSO, cocamidopropylbetaine, amidosulfobetaine-16, lauryl-N,N-(dimethylammonio)butyrate,lauryl-N,N-(dimethyl)-glycinebetaine, hexadecyl phosphocholine,lauryldimethylamine N-oxide, lauryl-N,N-(dimethyl)-propanesulfonate,3-(1-pyridinio)-1-propanesulfonate,3-(4-tert-butyl-1-pyridinio)-1-propanesulfonate, and combinationsthereof. A solvent or solution may comprise a denaturing speciesincluding, but not limited to, acetic acid, trichloroacetic acid,sulfosalicylic acid, sodium bicarbonate, ethanol, ethylenediaminetetraacetic acid (EDTA), urea, guanidinium chloride, lithiumperchlorate, sodium dodecyl sulfate, 2-mercaptoethanol, dithiothreitol,and tris(2-carboxyethyl) phosphine (TCEP).

A pH buffering species, cationic species, anionic species, surfactantspecies, or denaturing species may be present in a solvent compositionat a concentration of at least about 0.0001M, 0.001M, 0.01M, 0.02M,0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M,0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M,1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M,2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M,4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M, 5.1M, 5.2M,5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6M, 7M, 8M, 9M or more than10M. Alternatively or additionally, a pH buffering species, cationicspecies, anionic species, surfactant species, or denaturing species maybe present in a solvent or solution at a concentration of no more thanabout 10 M, 9M, 8M, 7M, 6M, 5.9M, 5.8M, 5.7M, 5.6M, 5.5M, 5.4M, 5.3M,5.2M, 5.1M, 5.0M, 4.9M, 4.8M, 4.7M, 4.6M, 4.5M, 4.4M, 4.3M, 4.2M, 4.1M,4.0M, 3.9M, 3.8M, 3.7M, 3.6M, 3.5M, 3.4M, 3.3M, 3.2M, 3.1M, 3.0M, 2.9M,2.8M, 2.7M, 2.6M, 2.5M, 2.4M, 2.3M, 2.2M, 2.1M, 2.0M, 1.9M, 1.8M, 1.7M,1.6M, 1.5M, 1.4M, 1.3M, 1.2M, 1.1M, 1.0M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M,0.4M, 0.3M, 0.2M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M,0.02M, 0.01M, 0.001M, 0.001M, or less than about 0.001M.

A pH buffering species, cationic species, anionic species, surfactantspecies, or denaturing species may be present in a solvent compositionin a weight percentage of at least about 0.0001 weight percent (wt %),0.001 wt %, 0.002 wt %, 0.003 wt %, 0.004 wt %, 0.005 wt %, 0.006 wt %,0.007 wt %, 0.008 wt %, 0.009 wt %, 0.01 wt %, 0.02 wt %, 0.03 wt %,0.04 wt %, 0.05 wt %, 0.06 wt %, 0.07 wt %, 0.08 wt %, 0.09 wt %, 0.1 wt%, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6 wt %, 0.7 wt %, 0.8 wt %,0.9 wt %, 1.0 wt %, 1.1 wt %, 1.2 wt %, 1.3 wt %, 1.4 wt %, 1.5 wt %,1.6 wt %, 1.7 wt %, 1.8 wt %, 1.9 wt %, 2 wt %, 2.1 wt %, 2.2 wt %, 2.3wt %, 2.4 wt %, 2.5 wt %, 2.6 wt %, 2.7 wt %, 2.8 wt %, 2.9 wt %, 3 wt%, 3.1 wt %, 3.2 wt %, 3.3 wt %, 3.4 wt %, 3.5 wt %, 3.6 wt %, 3.7 wt %,3.8 wt %, 3.9 wt %, 4 wt %, 4.1 wt %, 4.2 wt %, 4.3 wt %, 4.4 wt %, 4.5wt %, 4.6 wt %, 4.7 wt %, 4.8 wt %, 4.9 wt %, 5 wt %, 6 wt %, 7 wt %, 8wt %, 9 wt %, 10 wt %, or more than 10 wt %. Alternatively oradditionally, a pH buffering species, cationic species, anionic species,surfactant species, or denaturing species may be present in a solvent orsolution in a weight percentage of no more than about 10 wt %, 9 wt %, 8wt %, 7 wt %, 6 wt %, 5 wt %, 4.9 wt %, 4.8 wt %, 4.7 wt %, 4.6 wt %,4.5 wt %, 4.4 wt %, 4.3 wt %, 4.2 wt %, 4.1 wt %, 4.0 wt %, 3.9 wt %,3.8 wt %, 3.7 wt %, 3.6 wt %, 3.5 wt %, 3.4 wt %, 3.3 wt %, 3.2 wt %,3.1 wt %, 3.0 wt %, 2.9 wt %, 2.8 wt %, 2.7 wt %, 2.6 wt %, 2.5 wt %,2.4 wt %, 2.3 wt %, 2.2 wt %, 2.1 wt %, 2.0 wt %, 1.9 wt %, 1.8 wt %,1.7 wt %, 1.6 wt %, 1.5 wt %, 1.4 wt %, 1.3 wt %, 1.2 wt %, 1.1 wt %,1.0 wt %, 0.9 wt %, 0.8 wt %, 0.7 wt %, 0.6 wt %, 0.5 wt %, 0.4 wt %,0.3 wt %, 0.2 wt %, 0.1 wt %, 0.09 wt %, 0.08 wt %, 0.07 wt %, 0.06 wt%, 0.05 wt %, 0.04 wt %, 0.03 wt %, 0.02 wt %, 0.01 wt %, 0.009 wt %,0.008 wt %, 0.007 wt %, 0.006 wt %, 0.005 wt %, 0.004 wt %, 0.003 wt %,0.002 wt %, 0.001 wt %, 0.0001 wt %, or less than 0.0001 wt %.

A solvent or solution, having a nucleic acid (e.g., a nucleic acidnanostructure, SNAP, a complex thereof, or a component thereof), orother composition set forth herein, may be formulated to have a pH at avalue or within a range of values. A solvent or solution may have a pHof at least about 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4,8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0,11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2,12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4,13.5, 13.6, 13.7, 13.8, 13.9, 14.0 or more than about 14.0.Alternatively or additionally, a solvent or solution may have a pH of nomore than about 14.0, 13.9, 13.8, 13.7, 13.6, 13.5, 13.4, 13.3, 13.2,13.1, 13.0, 12.9, 12.8, 12.7, 12.6, 12.5, 12.4, 12.3, 12.2, 12.1, 12.0,11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8,10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, 10.0, 9.9, 9.8, 9.7, 9.6, 9.5,9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1,8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7,6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3,5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9,3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5,2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1,1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0, or less than about0.

A nucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof), as set forth herein, may be formed ormodified at a particular temperature or temperature range. Thetemperature at which a nucleic acid is formed or modified may dependupon the components being used. For example, the addition ofoligonucleotides to a SNAP structure may be limited by the meltingtemperature of certain oligonucleotides. In another example, a SNAPcomponent that is conjugated by a click reaction may be added at abenign temperature, such as room temperature. In some configurations, anucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof) may be formed in a single-step reaction(i.e., combining all necessary components) that requires multipletemperature changes (e.g., a melting temperature followed by a nucleicacid annealing temperature followed by a conjugation reactiontemperature). In other configurations, a nucleic acid (e.g., a nucleicacid nanostructure, SNAP, a complex thereof, or a component thereof) maybe formed in multiple steps, each with a unique temperature profile. Anucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof) formation process may occur at atemperature of at least about −100° C., −90° C., −80° C., −70° C., −60°C., −50° C., −40° C., −30° C., −20° C., −10° C., −5° C., 0° C., 4° C.,10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C.,55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 90° C., or more than 90°C. Alternatively or additionally, a nucleic acid (e.g., a nucleic acidnanostructure, SNAP, a complex thereof, or a component thereof)formation process may occur at a temperature of no more than about 90°C., 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., 40°C., 35° C., 30° C., 25° C., 20° C., 15° C., 10° C., 4° C., 0° C., −10°C., −20° C., −30° C., −40° C., −50° C., −60° C., −70° C., −80° C., −90°C., 100° C., or less than −100° C.

A nucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof), as set forth herein, may be stored ina suitable storage medium (e.g., a storage buffer). A nucleic acid maybe stored at a temperature that keeps a storage medium in a liquidstate. A nucleic acid may be stored at a temperature that causes astorage medium to freeze into a solid state. A nucleic acid may bestored before or after an analyte (e.g., a polypeptide) has been coupledto the nucleic acid. A nucleic acid may be stored at a temperature inone or more of the ranges set forth above for formation of a nucleicacid nanostructure.

A nucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof), as set forth herein, may remain stableduring storage. Stability may be indicated by a nucleic acid activityafter storage relative to a pre-storage baseline, such as ability tocouple an analyte (e.g., a polypeptide), ability to couple with anothernucleic acid, or ability to associate with a surface or interface. Anucleic acid may be stabilized against aggregation or sedimentation bythe presence of a surfactant or detergent species. A nucleic acid may bestabilized against degradation, such as oxidation, by the presence ofanti-oxidants or radical scavengers. A SNAP may be stable when storedfor a period of at least about 1 hr, 6 hrs, 12 hrs, 1 day, 2 days, 3days, 1 wk, 2 wks, 3 wks, 4 wks, 1 mth, 2 mths, 3 mths, 4 mths, 5 mths,6 mths, 9 mths, 1 yr, 2 yrs, 3 yrs, 4 yrs, 5 yrs, 10 yrs, or more than10 yrs. Alternatively or additionally, a nucleic acid may be stable whenstored for a period of no more than about 10 yrs, 5 yrs, 4 yrs, 3 yrs, 2yrs, 1 yrs, 9 mths, 6 mths, 5 mths, 4 mths, 3 mths, 2 mths, 1 mth, 4wks, 3 wks, 2 wks, 1 wk, 3 days, 2 days, 1 day, 12 hrs, 6 hrs, 1 hr, orless than 1 hr.

A nucleic acid, as set forth herein, may be provided as a component of akit. A kit may comprise a nucleic acid, as set forth herein, that isconfigured to be coupled to an analyte of interest. A kit may beprovided with a nucleic acid, as set forth herein, or a pluralitythereof. A collection kit may be specific to a particular assay to beperformed on the sample. For example, a collection kit for a polypeptideassay may include polypeptide-specific reagents to protect and/orpreserve polypeptides within a sample. A collection kit may include oneor more sample vessels, one or more reagents, instructions for use ofthe sample collection kit and optionally intermediate sample vessels, asealant for the vessel(s), a label for the vessel(s) such as a barcodeor radio frequency identification device (RFID), or packaging fortransport and/or storage of the sample vessel(s). A kit may include oneor more reagents for any of a variety of purposes, including samplepreservation, sample stability, sample quality control, processingand/or purification, and sample storage. A kit may include reagents suchas buffers, acids, bases, solvents, denaturants, surfactants,detergents, reactants, labels (e.g., fluorophores, radiolabels),indicator dyes, enzymes, enzyme inhibitors, oxygen scavengers, waterscavengers, humectants, affinity reagents (e.g., antibodies), or othercapture agents (e.g., biotinylated particles). A kit may include one ormore reagents in liquid or solid form. A kit may include one or moreseparate reagents and/or internal standards that are added to a samplevessel before or after sample preparation. A kit may include one or morereagents and/or internal standards that are provided within a samplecollection vessel. For example, reagents and/or internal standards maybe provided in a crystallized or coated form on a surface of acollection vessel, or may be in a liquid solution within the collectionvessel. In some configurations, a kit may further comprise an array orsolid support, as set forth herein. An array or solid support may beprovided in a kit with one or more nucleic acids present within ordeposited on the array or solid support.

A kit for an assay or other process may be utilized according to aprovided set of instructions. The instructions may be directed to use ofnucleic acids in accordance with teachings set forth herein. A kit mayprovide instructions for coupling an analyte of interest to a nucleicacid, for example by a method as set forth herein. A kit may provideinstructions for depositing a nucleic acid, as set forth herein, or anucleic acid coupled to an analyte of interest, to an array or solidsupport, as set forth herein. A kit may be utilized by a technician orself-collecting subject. A technician utilizing a kit may bespecifically trained in the proper utilization of the kit. A kitprotocol may employ one or more intermediate steps before preparation ofa sample is complete. Intermediate steps during sample preparation maybe performed in a vessel or in a separate medium (provided with the kitor provided by the collector). For example, a blood sample may befractionated by a phlebotomist, with only the red blood cell or plasmafraction saved for preparation. A kit may include indicator dyes, litmusstrips, or other methods of confirming successful sample collectionand/or preparation. A kit may include a sealant (e.g., an adhesive orsticker) to ensure that a sample has not been tampered with or damagedduring storage or transport. A kit may include a label for sampletracking by the collector or the analysis facility. A label for a vesselmay include a serial number, RFID, bar code or QR code. A label for avessel may be pre-printed or pre-applied to a vessel, or may be placedby a collector.

Methods of Nucleic Acid Fabrication

Nucleic acids (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof) as described in the present disclosuremay be fabricated by a suitable method. Fabrication of a nucleic acidmay comprise one or more of the steps of: 1) providing a scaffoldnucleic acid strand that is configured to couple a plurality ofoligonucleotides; 2) providing a plurality of oligonucleotides that areconfigured to couple to the scaffold strand; 3) providing one or moreadditional oligonucleotides that are configured to couple to thescaffold nucleic acid strand or other oligonucleotides; 4) providing oneor more oligonucleotides that are configured to couple to the scaffoldnucleic acid strand and are further configured to couple to an analyte;5) providing one or more oligonucleotides that are configured to coupleto the scaffold nucleic acid strand and are coupled to an analyte; 6)providing one or more oligonucleotides that are configured to couple tothe scaffold nucleic acid strand and are further configured to couple toa surface; 7) annealing the scaffold nucleic acid strand to a pluralityof oligonucleotides to form a SNAP; 8) annealing the scaffold strand toan oligonucleotide that is configured to couple to an analyte; 9)annealing the scaffold strand to an oligonucleotide that is coupled toan analyte; 10) annealing the scaffold strand to an oligonucleotide thatis configured to couple to a surface; and 11) forming one or morecouplings or cross-links between two or more oligonucleotides of thenucleic acid.

Fabrication of detectable probes comprising nucleic acid retainingcomponents (e.g., DNA origami, DNA nanoballs) may be formed byconventional techniques. DNA nanoballs may be fabricated by a methodsuch as rolling circle amplification to generate a scaffold strand thatmay be further modified to couple or conjugate a plurality of bindingcomponents and/or detectable labels. Exemplary methods for makingnucleic acid nanoballs are described, for example, in U.S. Pat. No.8,445,194, which is incorporated herein by reference. Nucleic acidretaining components comprising sections of double-stranded DNA (e.g.,DNA origami) may be fabricated, for example, using techniques describedin Rothemund, Nature 440:297-302 (2006) and U.S. Pat. Nos. 8,501,923 and9,340,416, each of which is incorporated herein by reference. Aretaining component may be formed by a scaffold strand that ishybridized with additional oligonucleotides.

FIG. 36A shows a first pathway to forming a SNAP comprising a DNAorigami that is coupled to a plurality of analytes and a plurality ofdetectable labels. Oligonucleotides with coupled or conjugated analytes3620 and oligonucleotides with conjugated detectable labels 3630 areprepared before the retaining component is assembled. Theoligonucleotides with conjugated binding components 3620 andoligonucleotides with coupled or conjugated detectable labels 3630 arecontacted with a single-stranded scaffold 3610 (e.g., M13 phage DNA,single-stranded plasmid DNA) and additional structural nucleic acids3640. The nucleic acids are contacted in a suitable DNA buffer at anelevated temperature (e.g., at least about 50° C., 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., 90° C., or about 95° C.), thencooled. Oligonucleotides will hybridize to the scaffold strand 3610 atthe appropriate sequence-dependent positions to form a SNAP-analyteconjugate 3650. The number of analytes coupled to a SNAP may becontrolled by using fewer or greater numbers of oligonucleotides thatare coupled to analytes or are configured to be coupled to analytes, orby altering a sequence of a scaffold strand.

FIG. 36B shows an alternative pathway to forming a SNAP with a pluralityof coupled analytes and a plurality of detectable labels.Oligonucleotides with handles that are configured to couple or conjugateanalytes 3625 and oligonucleotides with moieties that are configured tocouple or conjugate detectable labels 3635 are prepared before theretaining component is assembled. The oligonucleotides with moietiesthat are configured to conjugate analytes 3625 and oligonucleotides withmoieties that are configured to couple or conjugate detectable labels3635 are contacted with a single-stranded scaffold 3610 (e.g., M13 phageDNA, plasmid DNA) and additional structural nucleic acids 3640. Thenucleic acids are contacted in a suitable DNA buffer at an elevatedtemperature (e.g., at least about 50° C., 55° C., 60° C., 65° C., 70°C., 75° C., 80° C., 85° C., 90° C., or about 95° C.), then cooled. Aftercooling, a SNAP 3655 that is configured to bind a plurality of analytesand/or label components is formed. The retaining component 3655 iscontacted with a plurality of analytes 3628 and/or label components 3638that have complementary moieties to the moieties on the SNAP 3655 in asuitable conjugation buffer. After coupling or conjugation of theplurality of analytes 3628 and/or the plurality of label components3638, a SNAP-analyte conjugate 3650 is formed.

In some configurations, a detectable nucleic acid (e.g., a nucleic acidnanostructure, a SNAP), as set forth herein, may be formed by thecoupling or conjugation of an analyte and/or a label component by thereaction of a reactive group configured to form a bond with anothermolecule or group, e.g., a bioorthogonal reaction or click-typechemistry (see, for example, U.S. Pat. Nos. 6,737,236 and 7,427,678,each incorporated herein by reference in its entirety); azide alkyneHuisgen cycloaddition reactions, which use a copper catalyst (see, forexample, U.S. Pat. Nos. 7,375,234 and 7,763,736, each incorporatedherein by reference in its entirety); Copper-free Huisgen reactions(“metal-free click”) using strained alkynes or triazine-hydrazinemoieties which can link to aldehyde moieties (see, for example, U.S.Pat. No. 7,259,258, which is incorporated by reference); triazinechloride moieties which can link to amine moieties; carboxylic acidmoieties which can link to amine moieties using a coupling reagent, suchas EDC; thiol moieties which can link to thiol moieties; alkene moietieswhich can link to dialkene moieties that are coupled through Diels-Alderreactions; and acetyl bromide moieties which can link to thiophosphatemoieties (see, for example, WO 2005/065814, which is incorporated byreference). A reactive handle may comprise a functional group that isconfigured to react via a click reaction (e.g., metal-catalyzedazide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition,strain-promoted azide-nitrone cycloaddition, strained alkene reactions,thiol-ene reaction, Diels-Alder reaction, inverse electron demandDiels-Alder reaction, [3+2] cycloaddition, [4+1] cycloaddition,nucleophilic substitution, dihydroxylation, thiol-yne reaction,photoclick, nitrone dipole cycloaddition, norbornene cycloaddition,oxanobornadiene cycloaddition, tetrazine ligation, tetrazole photoclickreactions). Exemplary silane-derivative CLICK-type reactants may includealkenes, alkynes, azides, epoxides, amines, thiols, nitrones,isonitriles, isocyanides, aziridines, activated esters, and tetrazines(e.g., dibenzocyclooctyne-azide, methyltetrazine-transcyclooctylene,epoxide-thiol, etc.). A click-type reaction may provide an advantageousmethod of rapidly forming a bond under benign conditions (e.g., roomtemperature, aqueous solvents). In some configurations, a SNAP maycomprise cross-linking molecules that are form bonds that irreversiblycouple a first SNAP component to a second SNAP component. Cross-linkingmolecules may include chemical cross-linking molecules andphoto-initiated cross-linking molecules.

In some configurations, a nucleic acid or other component of a nucleicacid may include different species of reactive groups. The use ofdifferent reactive groups can provide a level of control over the numberand location of different components that will be coupled or conjugatedto the nucleic acid. In particular configurations the different reactivegroups demonstrate orthogonal reactivity, whereby a first component hasa moiety that is reactive for a first reactive handle (i.e. reactivemoiety) on the probe but not substantially reactive with a secondreactive handle on the probe, and whereby a second component has amoiety that is reactive for the second reactive handle but not the firstreactive handle. Accordingly, the number of different analytes and theirlocations can be adjusted by appropriate use of orthogonal reactivehandles on a detectable probe or the number of different labelcomponents and their locations can be adjusted by appropriate use oforthogonal reactive handles on a detectable probe. Moreover, analytescan be located differently from label components on a nucleic acid byappropriate use of orthogonal reactive handles, respectively, on thenucleic acid.

Following synthesis of a nucleic acid (e.g., a nucleic acidnanostructure, SNAP, a complex thereof, or a component thereof), as setforth herein, formed structures may be purified by one or moreadditional processes. A nucleic acid may undergo one or more separationprocesses to remove unwanted components, such as one or more of: 1)uncoupled oligonucleotides; 2) uncoupled analytes; 3) uncoupledmodifying groups; 4) buffer components; 5) partially-formed nucleicacids; 6) misformed nucleic acids; and 7) excess nucleic acids. Anucleic acid may undergo a dilution or concentration process to adjust aconcentration of a nucleic acid containing solution. Nucleic acids maybe separated from unwanted components by any suitable method, includingwithout limitation, for example high-pressure liquid chromatography(HPLC), size-exclusion chromatography (SEC), affinity chromatography,ultracentrifugation, osmosis, reverse osmosis, and ultrafiltration. Insome configurations, a separation may be performed on a separationmedium (e.g., a chromatography column) that is not specified for nucleicacids separation. In some configurations, a separation may be performedon a separation medium (e.g., a chromatography column) that is notspecified for the expected hydrodynamic size range of the separatednucleic acids.

Polypeptide Assays

The present disclosure provides systems, compositions, and methods forforming particles that are useful for coupling single analytes. Thepresent disclosure further provides systems, compositions, and methodsfor forming single-analyte arrays that are useful when performingvarious single-analyte assays, including assays of biological analytes(e.g., genomics, transcriptomics, proteomics, metabolomics, etc.) andnon-biological analytes (e.g., carbon nanoparticles, inorganicnanoparticles, etc.). In some configurations, the providedsingle-analyte arrays may be especially useful for single-polypeptideproteomic assays such as, for example affinity reagent-basedcharacterization assays (e.g., fluorescence-based or barcode-basedaffinity binding characterizations) or peptide sequencing assays (e.g.,Edman-type degradation fluorosequencing or affinity reagent-basedassays).

The present disclosure further provides methods for detecting one ormore polypeptide (e.g., sample polypeptide, standard polypeptide etc.)or polypeptide product (e.g. sample polypeptide composite, standardpolypeptide composite, etc.). A polypeptide can be detected using one ormore probes having known binding affinity for the polypeptide. The probeand/or the polypeptide can be bound to form a complex and then formationof the complex can be detected. The complex can be detected directly,for example, due to a label that is present on the probe or polypeptide.In some configurations the complex need not be directly detected, forexample, in formats where the complex is formed and then the probe,polypeptide, or a tag or label component that was present in the complexis then detected.

In some detection assays, a protein can be cyclically modified and themodified products from individual cycles can be detected. In someconfigurations, a protein can be sequenced by a sequential process inwhich each cycle includes steps of labeling and removing the aminoterminal amino acid of a protein and detecting the label. Accordingly, amethod of detecting a protein can include steps of (i) exposing aterminal amino acid on the protein; (ii) detecting a change in signalfrom the protein; and (iii) identifying the type of amino acid that wasremoved based on the change detected in step (ii). The terminal aminoacid can be exposed, for example, by removal of one or more amino acidsfrom the amino terminus or carboxyl terminus of the protein. Steps (i)through (iii) can be repeated to produce a series of signal changes thatis indicative of the sequence for the protein.

In a first configuration of the above method, one or more types of aminoacids in the protein can be attached to a label that uniquely identifiesthe type of amino acid. In this configuration, the change in signal thatidentifies the amino acid can be loss of signal from the respectivelabel. Exemplary compositions and techniques that can be used to removeamino acids from a protein and detect signal changes are those set forthin Swaminathan et al., Nature Biotech. 36:1076-1082 (2018); or U.S. Pat.Nos. 9,625,469 or 10,545,153, each of which is incorporated herein byreference. Methods and apparatus under development by Erisyon, Inc.(Austin, Tex.) may also be useful for detecting proteins.

In a second configuration of the above method, the terminal amino acidof the protein can be recognized by an affinity agent that is specificfor the terminal amino acid or specific for a label moiety that ispresent on the terminal amino acid. The affinity agent can be detectedon the array, for example, due to a label on the affinity agent.Optionally, the label is a nucleic acid barcode sequence that is addedto a primer nucleic acid upon formation of a complex. The formation ofthe complex and identity of the terminal amino acid can be determined bydecoding the barcode sequence. Exemplary affinity agents and detectionmethods are set forth in US Pat. App. Pub. No. 2019/0145982 A1;2020/0348308 A1; or 2020/0348307 A1, each of which is incorporatedherein by reference. Methods and apparatus under development by Encodia,Inc. (San Diego, Calif.) may also be useful for detecting proteins.

Cyclical removal of terminal amino acids from a protein can be carriedout using an Edman-type sequencing reaction in which a phenylisothiocyanate reacts with a N-terminal amino group under mildlyalkaline conditions (e.g., about pH 8) to form a cyclicalphenylthiocarbamoyl Edman complex derivative. The phenyl isothiocyanatemay be substituted or unsubstituted with one or more functional groups,linker groups, or linker groups containing functional groups. AnEdman-type sequencing reaction can include variations to reagents andconditions that yield a detectable removal of amino acids from a proteinterminus, thereby facilitating determination of the amino acid sequencefor a protein or portion thereof. For example, the phenyl group can bereplaced with at least one aromatic, heteroaromatic or aliphatic groupwhich may participate in an Edman-type sequencing reaction, non-limitingexamples including: pyridine, pyrimidine, pyrazine, pyridazoline, fusedaromatic groups such as naphthalene and quinoline), methyl or otheralkyl groups or alkyl group derivatives (e.g., alkenyl, alkynyl,cyclo-alkyl). Under certain conditions, for example, acidic conditionsof about pH 2, derivatized terminal amino acids may be cleaved, forexample, as a thiazolinone derivative. The thiazolinone amino acidderivative under acidic conditions may form a more stablephenylthiohydantoin (PTH) or similar amino acid derivative which can bedetected. This procedure can be repeated iteratively for residualprotein to identify the subsequent N-terminal amino acid. Manyvariations of Edman-type degradation have been described and may be usedincluding, for example, a one-step removal of an N-terminal amino acidusing alkaline conditions (Chang, J.Y., FEBS LETTS., 1978, 91(1),63-68). In some cases, Edman-type reactions may be thwarted byN-terminal modifications which may be selectively removed, for example,N-terminal acetylation or formylation (e.g., see Gheorghe M.T., BergmanT. (1995) in Methods in Protein Structure Analysis, Chapter 8:Deacetylation and internal cleavage of Proteins for N-terminal SequenceAnalysis. Springer, Boston, Mass.

Non-limiting examples of functional groups for substituted phenylisothiocyanate may include ligands (e.g. biotin and biotin analogs) forknown receptors, labels such as luminophores, or reactive groups such asclick functionalities (e.g. compositions having an azide or acetylenemoiety). The functional group may be a DNA, RNA, peptide or smallmolecule barcode or other tag which may be further processed and/ordetected.

The removal of an amino terminal amino acid using Edman-type processesutilizes at least two main steps, the first step includes reacting anisothiocyanate or equivalent with protein N-terminal residues to form arelatively stable Edman complex, for example, a phenylthiocarbamoylcomplex. The second step includes removing the derivatized N-terminalamino acid, for example, via heating. The protein, now having beenshortened by one amino acid, may be detected, for example, by contactingthe protein with a labeled affinity agent that is complementary to theamino terminus and examining the protein for binding to the agent, or bydetecting loss of a label that was attached to the removed amino acid.

Edman-type processes can be carried out in a multiplex format to detect,characterize or identify a plurality of proteins. A method of detectinga protein can include steps of (i) exposing a terminal amino acid on aprotein at an address of an array; (ii) binding an affinity agent to theterminal amino acid, where the affinity agent comprises a nucleic acidtag, and where a primer nucleic acid is present at the address; (iii)extending the primer nucleic acid, thereby producing an extended primerhaving a copy of the tag; and (iv) detecting the tag of the extendedprimer. The terminal amino acid can be exposed, for example, by removalof one or more amino acids from the amino terminus or carboxyl terminusof the protein. Steps (i) through (iv) can be repeated to produce aseries of tags that is indicative of the sequence for the protein. Themethod can be applied to a plurality of proteins on the array and inparallel. Whatever the plexity, the extending of the primer can becarried out, for example, by polymerase-based extension of the primer,using the nucleic acid tag as a template. Alternatively, the extendingof the primer can be carried out, for example, by ligase- orchemical-based ligation of the primer to a nucleic acid that ishybridized to the nucleic acid tag. The nucleic acid tag can be detectedvia hybridization to nucleic acid probes (e.g., in an array),amplification-based detections (e.g. PCR-based detection, or rollingcircle amplification-based detection) or nuclei acid sequencing (e.g.cyclical reversible terminator methods, nanopore methods, or singlemolecule, real time detection methods). Exemplary methods that can beused for detecting proteins using nucleic acid tags are set forth in USPat. App. Pub. No. 2019/0145982 A1; 2020/0348308 A1; or 2020/0348307 A1,each of which is incorporated herein by reference.

Polypeptides can also be detected based on their enzymatic or otherbiological activity. For example, a polypeptide can be contacted with areactant that is converted to a detectable product by an enzymaticactivity of the polypeptide. In other assay formats, a first polypeptidehaving a known enzymatic function can be contacted with a secondpolypeptide to determine if the second polypeptide changes the enzymaticfunction of the first polypeptide. As such, the first polypeptide servesas a reporter system for detection of the second polypeptide. Exemplarychanges that can be observed include, but are not limited to, activationof the enzymatic function, inhibition of the enzymatic function,degradation of the first polypeptide or competition for a reactant orcofactor used by the first polypeptide.

The presence or absence of post-translational modifications (PTM) can bedetected using a composition, apparatus or method set forth herein. APTM can be detected using an affinity agent that recognizes the PTM orbased on a chemical property of the PTM. Exemplary PTMs that can bedetected, identified or characterized include, but are not limited to,myristoylation, palmitoylation, isoprenylation, prenylation,farnesylation, geranylgeranylation, lipoylation, flavin moietyattachment, Heme C attachment, phosphopantetheinylation, retinylideneSchiff base formation, dipthamide formation, ethanolaminephosphoglycerol attachment, hypusine, beta-Lysine addition, acylation,acetylation, deacetylation, formylation, alkylation, methylation,C-terminal amidation, arginylation, polyglutamylation, polyglyclyation,butyrylation, gamma-carboxylation, glycosylation, glycation,polysialylation, malonylation, hydroxylation, iodination, nucleotideaddition, phosphoate ester formation, phosphoramidate formation,phosphorylation, adenylylation, uridylylation, propionylation,pyrolglutamate formation, S-glutathionylation, S-nitrosylation,S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation,sulfation, glycation, carbamylation, carbonylation, isopeptide bondformation, biotinylation, carbamylation, oxidation, reduction,pegylation, ISGylation, SUMOylation, ubiquitination, neddylation,pupylation, citrullination, deamidation, elminylation, disulfide bridgeformation, proteolytic cleavage, isoaspartate formation, racemization,and protein splicing.

PTMs may occur at particular amino acid residues of a protein. Forexample, the phosphate moiety of a particular proteoform can be presenton a serine, threonine, tyrosine, histidine, cysteine, lysine, aspartateor glutamate residue of the protein. In other examples, an acetyl moietycan be present on the N-terminus or on a lysine; a serine or threonineresidue can have an O-linked glycosyl moiety; an asparagine residue canhave an N-linked glycosyl moiety; a proline, lysine, asparagine,aspartate or histidine amino acid can be hydroxylated; an arginine orlysine residue can be methylated; or the N-terminal methionine or at alysine amino acid can be ubiquitinated.

Polypeptides can also be detected based on their binding interactionswith other molecules such as polypeptides (e.g., with or without posttranslational modifications), nucleic acids, nucleotides, metabolites,small molecules that participate in biological signal transductionpathways, biological receptors or the like. For example, a polypeptidethat participates in a signal transduction pathway can be identified bydetecting binding of the polypeptide with a second polypeptide that isknown to be its binding partner in the pathway. Generally, a targetpolypeptide can be conjugated to a SNAP or SNAP complex and thencontacted with a probe polypeptide, or other probe molecule, that isknown to have affinity for the polypeptide. The target polypeptide canbe identified based on observed binding by the probe molecule or lack ofbinding by the probe molecule. The probe molecule can optionally belabeled using labels set forth herein or known in the art.

In some configurations of the polypeptide detection methods set forthherein, the polypeptides can be detected on a solid support. Forexample, polypeptides can be attached to a support, the support can becontacted with probes in solution, the probes can interact with thepolypeptides, thereby producing a detectable signal, and then the signalcan be detected to determine the presence of the polypeptides. Inmultiplexed versions of this approach, different polypeptides can beattached to different addresses in an array, and the probing anddetection steps can occur in parallel. In another example, probes can beattached to a solid support, the support can be contacted withpolypeptides in solution, the polypeptides can interact with the probes,thereby producing a detectable signal, and then the signal can bedetected to determine the presence of the polypeptides. This approachcan also be multiplexed by attaching different probes to differentaddresses of an array. Polypeptides can be attached to a support viaconjugation to SNAPs or SNAP complexes. For example, a plurality ofpolypeptides can be conjugated to a plurality of SNAPs or SNAPcomplexes, such that each polypeptide-conjugated SNAP or SNAP complexforms an address in the array. In yet another approach, polypeptides canbe detected using mass spectrometry methods. Several exemplary detectionmethods are set forth below and elsewhere herein. It will be understoodthat other detection methods can also be used.

Typical polypeptide detection methods, such as enzyme linkedimmunosorbent assay (ELISA), achieve high-confidence characterization ofone or more polypeptide in a sample by exploiting high specificitybinding of antibodies, aptamers or other binding reagents to thepolypeptide(s) and detecting the binding event while ignoring all otherpolypeptides in the sample. ELISA is generally carried out at low plexscale (e.g. from one to several hundred different polypeptides detectedin parallel or in succession) but can be used at higher plexity. One ormore polypeptides can be conjugated to one or more SNAPs or SNAPcomplexes and the conjugated polypeptide(s) can be detected using ELISA.

ELISA methods can be carried out by detecting immobilized bindingreagents and/or polypeptides in multiwell plates, detecting immobilizedbinding reagents and/or polypeptides on arrays, or detecting immobilizedbinding reagents and/or polypeptides on particles in microfluidicdevices. Exemplary plate-based methods include, for example, theMULTI-ARRAY technology commercialized by MesoScale Diagnostics(Rockville, Md.) or Simple Plex technology commercialized by ProteinSimple (San Jose, Calif.). Exemplary, array-based methods include, butare not limited to those utilizing Simoa® Planar Array Technology orSimoa® Bead Technology, commercialized by Quanterix (Billerica, Mass.).Further exemplary array-based methods are set forth in U.S. Pat. Nos.9,678,068; 9,395,359; 8,415,171; 8,236,574; or 8,222,047, each of whichis incorporated herein by reference. Exemplary microfluidic detectionmethods include those commercialized by Luminex (Austin, Tex.) under thetrade name xMAP® technology or used on platforms identified as MAGPIX®,LUMINEX® 100/200 or FEXMAP 3D®. Plate-based methods of microfluidicdetection methods can be modified to use SNAPs or SNAP complexes as setforth herein.

Other detection methods that can also be used, and that are particularlyuseful at low plex scale include procedures that employ SOMAmer reagentsand SOMAscan assays commercialized by Soma Logic (Boulder, Colo.). Inone configuration, a sample is contacted with aptamers that are capableof binding polypeptides with high specificity for the amino acidsequence of the polypeptides. The resulting aptamer-polypeptidecomplexes can be separated from other sample components, for example, byattaching the complexes to beads, SNAPs or SNAP complexes that areremoved from the sample. The aptamers can then be isolated and, becausethe aptamers are nucleic acids, the aptamers can be detected using anyof a variety of methods known in the art for detecting nucleic acids,including for example, hybridization to nucleic acid arrays, PCR-baseddetection, or nucleic acid sequencing. Exemplary methods andcompositions for use in an aptamer-based or other detection method setforth herein are set forth in U.S. Pat. Nos. 8,404,830; 8,975,388;9,163,056; 9,938,314; 10,239,908; 10,316,321 or 10,221,207. Furtherexamples are set forth in U.S. Pat. Nos. 7,855,054; 7,964,356;8,975,026; 8,945,830; 9,404,919; 9,926,566; 10,221,421; 10,316,321 or10,392,621. The above patents are incorporated herein by reference. Theaptamers or polypeptides set forth above or in the above references canbe attached to SNAPs or SNAP complexes as set forth herein.

Polypeptides can also be detected based on proximity of two or moreprobes. For example, two probes can each include a receptor componentand a nucleic acid component. When the probes bind in proximity to eachother, for example, due to ligands for the respective receptors being ona single polypeptide, or due to the ligands being present on twopolypeptides that associate with each other, the nucleic acids caninteract to cause a modification that is indicative of the proximity.For example, one of the nucleic acids can be extended using the othernucleic acid as a template, one of the nucleic acids can form a templatethat positions the other nucleic acid for ligation to another nucleicacid, or the like. Exemplary methods are commercialized by OlinkProteomics AB (Uppsala Sweden) or set forth in U.S. Pat. Nos. 7,306,904;7,351,528; 8,013,134; 8,268,554 or 9,777,315, each of which isincorporated herein by reference. The polypeptides, probes, ligands orreceptors set forth above or in the above references can be attached toa nucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof) as set forth herein.

A method of detecting a polypeptide, can include a step of detecting asample polypeptide (e.g. a sample polypeptide conjugate) and/ordetecting a standard polypeptide (e.g. a standard polypeptideconjugate). In one configuration, detection can include steps of (i)contacting a first set of binding reagents with a sample polypeptide,and/or a standard polypeptide, and (ii) detecting binding of the samplepolypeptide and/or standard polypeptide to a binding reagent in thesecond set of binding reagents. The method can optionally include one ormore of the further steps of (iii) removing the first set of bindingreagents, (iv) binding a second set of binding reagents to the samplepolypeptide, and/or the standard polypeptide, where binding reagents inthe second set are different from binding reagents in the first set, and(v) detecting binding of the sample polypeptide and/or standardpolypeptide to a binding reagent in the second set of binding reagents.The method can optionally be carried out for one or more samplepolypeptides in an array or standard polypeptides. Methods and apparatusthat employ standard polypeptides are set forth in U.S. Pat. App. Ser.No. 63/139,818, which is incorporated herein by reference. The samplepolypeptides or standard polypeptides set forth above or in the abovereference can be attached to a nucleic acid (e.g., a nucleic acidnanostructure, SNAP, a complex thereof, or a component thereof) as setforth herein.

High specificity binding reagents can be useful in a number ofpolypeptide detection methods. Alternatively, detection can be based onmultiple low specificity detection cycles that are performed on a samplesuch that the individual cycles may detect multiple polypeptides whilenot necessarily distinguishing one of the detected polypeptides fromanother in any one of the cycles. However, using compositions andmethods set forth herein, results from multiple cycles can be combinedto achieve high-confidence quantification, identification orcharacterizations of a plurality of individual polypeptides in thesample. In many embodiments, one or more of the individual cycles yieldambiguous results with regard to distinguishing the identity of a subsetof polypeptides that produce detectable signal; however, characterizingthe signals across the multiple cycles allows individual polypeptides tobe individually and unambiguously identified. The resulting set ofidentified polypeptides can be larger than the number of polypeptidesthat produce signal from any of the individual cycles.

Some configurations of detection methods that are based on multiple lowspecificity detection cycles may be understood, to some extent, viaanalogies to the children's game “20 Questions.” An objective of thisgame is to identify a target answer in as few questions as possible. Aneffective tactic is to ask questions on characteristics ranging frombroad characteristics (e.g., “Is it a person, place, or thing?”, “Is theperson in this room?”) to narrow characteristics (e.g., “Is the personnamed ‘Keith’?”). In general, it is possible to identify a character inthe game by asking substantially fewer questions (N) than the possiblenumber of answers (M), i.e. N<<M. By analogy, affinity reagents used insome configurations of the detection methods set forth herein, may havea broad range of interactions with respect to a population ofpolypeptides. For example, an affinity reagent may be considered to be a‘promiscuous’ affinity reagent due to its affinity for a single epitopethat is present in a plurality of different polypeptides in a sample, ordue to its affinity for a plurality of different epitopes that arepresent in one or more polypeptides in the sample. By testing for theinteraction of an affinity reagent with a polypeptide, information isacquired regardless of whether an interaction is observed. For example,a failure of an affinity reagent to bind a polypeptide is indicative ofthe polypeptide lacking the epitope for the affinity reagent.

In the above-described analogy of 20 Questions, the outcome is basedupon clear articulation of queries and answers, and is also based uponaccurate and reliable answers (e.g., type, size, attributes, etc.). Byanalogy, polypeptide characterization by the measurement of affinityreagent interactions may be more difficult when the measurements areprone to a degree of systematic or random error or uncertainty. Forexample, measurement accuracy of affinity reagent (e.g., antibody)interactions with binding targets (e.g. epitopes) may be affected bynumerous factors such as system detection limits or sensitivity,non-specific interactions between epitopes and affinity reagents (falsepositives), or stochastic, time-dependent reversal of an interaction(false negatives).

It is not uncommon for polypeptide characterization measurements tocontain a degree of uncertainty. High-confidence characterization may beachieved by utilizing multiple low specificity detection cycles incombination with a probabilistic decoding approach. The overlaying orcombining of binary polypeptide interaction data (e.g., affinity reagentA1, which interacts with epitope X, was not observed to interact withunknown polypeptide P, therefore, polypeptide P does not contain epitopeX) may lead to improper polypeptide characterization due to theinclusion or exclusion of possible candidate states due to measurementerror. By contrast, overlaying or combining probabilistic polypeptideinteraction data may permit an algorithm to converge to ahigh-confidence prediction of polypeptide identity without needing toexclude any candidate states. For example, if affinity reagents A1 to A6are known to interact with a known polypeptide P1 with interactionprobabilities, and measurable interactions of affinity reagents A2, A5and A6 are observed against an unknown polypeptide P, it may beconcluded that polypeptide P is likely not polypeptide P1 (2 of 3 likelyinteractions were not observed; 2 of 3 unlikely interactions wereobserved). Moreover, a probability-based characterization may beassigned a degree of confidence such that a prediction for each observedpolypeptide may be made when the degree of confidence rises above athreshold degree of confidence. For example, in the above observation ofpolypeptide P, the six described observations need not provide a highenough degree of confidence to eliminate polypeptide P1 as a possibleidentity, but similar trends over 20 or more affinity reagents mayprovide sufficient degree of confidence to eliminate P1 as a possibleidentity. Accordingly, polypeptide P1 can be subjected to bindingreactions with a series of promiscuous affinity reagents, and althoughthe observation from each binding reaction taken individually may beambiguous with regard to identifying the polypeptide, decoding theobservations from the series of binding reactions may identifypolypeptide P1 with an acceptable level of confidence.

A polypeptide detection assay that is based on multiple low specificitydetection cycles may be configured to permit polypeptidecharacterization at an individual or single-molecule level. Polypeptidesto be characterized may be provided on a solid support containingunique, detectably resolvable characterization sites. For example, thepolypeptides can be attached to the sites via conjugation to a nucleicacid (e.g., a nucleic acid nanostructure, SNAP, a complex thereof, or acomponent thereof). Such characterization sites may be spaced, arrayed,or otherwise ordered to allow individual sites to be distinguished onefrom another when detecting their interactions with affinity reagents. Asolid support may comprise a sufficient number of unique, opticallyresolvable characterization sites to accommodate a plurality, majority,or all polypeptides from a sample, such as at least about 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or more than 1×10¹²sites. Each site may contain a known number of polypeptides that are tobe characterized. In some cases, a characterization site may contain asingle polypeptide molecule to be detected, identified or characterized.In other cases, a site may contain multiple polypeptide molecules, withat least one molecule to be detected. For example, the polypeptidemolecule to be detected can be one subunit in a larger protein havingmultiple different subunits.

In some cases, polypeptide detection assays that are based on multiplelow specificity detection cycles may utilize affinity reagents such asantibodies (or functional fragments thereof), aptamers, mini proteinbinders, or any other suitable binding reagent. Affinity reagents may bepromiscuous affinity reagents that possess a likelihood to interact with(e.g., bind to) more than one polypeptide in a sample. In some cases,the affinity reagents may possess a likelihood to interact with two ormore unique, structurally dissimilar proteins in a sample. For example,an affinity reagent may bind with near-equal probability to a particularmembrane protein and a particular cytoplasmic protein based upon aregion of structural similarity. In some cases, a binding affinityreagent may possess a likelihood of binding to a particular amino acidepitope or family of epitopes regardless of the sequence context (e.g.,amino acid sequence upchain and/or downchain from the epitope). Anaffinity reagent can bind to a polypeptide that is conjugated to anucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof).

An affinity reagent that is used for multiple low specificity detectioncycles may be characterized such that it has an identified, determined,or assessed probability-based binding profile. An affinity reagent mayhave the property of binding to a first polypeptide with an identified,determined, or assessed binding probability of greater than about 50%(e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, 99.99%,99.999% or greater than about 99.999%) and binding to a secondstructurally non-identical polypeptide with an identified, determined,or assessed binding probability of less than about 50% (e.g., no morethan about 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or lessthan about 0.001%). In a particular case, the difference in observedbinding probabilities of the affinity reagent to the first and secondpolypeptides may be due to the presence, absence, or inaccessibility ofa particular epitope or family of epitopes in either the first or secondpolypeptide. Probabilistic affinity reagent binding profiles may bedetermined or identified by in vitro measurements or in silicopredictions.

Polypeptide detection methods that are based on multiple low specificitydetection cycles may further incorporate computational decodingapproaches that are optimized for the above-described affinity reagents.The decoding approaches may overlay or combine data from multiple roundsof detecting affinity reagent interaction with individual polypeptides,and can assign a degree of confidence for detection of signal from eachpolypeptide. For example, affinity reagent interactions can be detectedfor each site in an array of sites, and a degree of confidence can beassigned to detection of each signal at each site. Similarly, a degreeof confidence can be assigned to a series of detection events at eachsite. A polypeptide may be considered identified or characterized if thedegree of confidence for a prediction based upon overlayed or combinedaffinity reagent interaction data exceeds a threshold degree ofconfidence. The threshold degree of confidence for a polypeptidecharacterization prediction may depend upon the nature of thecharacterization. The threshold degree of confidence may fall in a rangefrom about 50% to about 99.999%, such as about 50%, 60%, 70%, 80%, 90%,95%, 99%, 99.99%, or about 99.999%. In some cases, the threshold degreeof confidence may be outside this range. In some cases, thecomputational decoding approaches may incorporate machine learning ortraining algorithms to update or refine the determined or identifiedprobabilistic interaction profile for the affinity reagents orpolypeptides with increased information or in ever widening contexts.

Particularly useful methods and algorithms that can be used fordetection methods employing multiple low specificity detection cyclesare set forth, for example, in U.S. Pat. No. 10,473,654; or PCTPublication No. WO 2019/236749 A2; or US Pat. App. Pub. Nos.2020/0082914 A1 or 2020/0090785 A1, each of which is incorporated hereinby reference. The methods set forth above and in the precedingreferences can be modified to use SNAPs or SNAP complexes of the presentdisclosure, for example, to attach polypeptides to a solid support.

A method of detecting a polypeptide, can include a process of detectinga sample polypeptide, the process including steps of (i) binding a firstbinding reagent to a sample polypeptide at an address of an array, wherethe binding reagent comprises a nucleic acid tag, and where a primernucleic acid is present at the address; (ii) extending the primernucleic acid, thereby producing an extended primer having a copy of thetag; and (iii) detecting the tag of the extended primer. The polypeptidecan be attached at the address of the array via conjugation to a nucleicacid (e.g., a nucleic acid nanostructure, SNAP, a complex thereof, or acomponent thereof). The extending of the primer can be carried out, forexample, by polymerase-based extension of the primer, using the nucleicacid tag as a template. Alternatively, the extending of the primer canbe carried out, for example, by ligase or chemical based ligation of theprimer to a nucleic acid that is hybridized to the nucleic acid tag. Thenucleic acid tag can be detected via hybridization to nucleic acidprobes (e.g., in a microarray), amplification-based detections (e.g.PCR-based detection, or rolling circle amplification-based detection) ornucleic acid sequencing (e.g. cyclical reversible terminator methods,nanopore methods, or single molecule, real time detection methods).Exemplary methods that can be used for detecting polypeptides usingnucleic acid tags are set forth in US Pat. App. Pub. No. 2019/0145982A1; 2020/0348308 A1; or 2020/0348307 A1, each of which is incorporatedherein by reference.

A method of detecting a polypeptide, can include a process of detectinga sample polypeptide, the process including steps of (i) exposing aterminal amino acid on the polypeptide; (ii) detecting a change insignal from the polypeptide; and (iii) identifying the type of aminoacid that was removed based on the change detected in step (ii). Theterminal amino acid can be exposed, for example, by removal of one ormore amino acids from the amino terminus or carboxyl terminus of thepolypeptide. Steps (i) through (iii) can be repeated to produce a seriesof signal changes that is indicative of the sequence for thepolypeptide. Optionally, one or more different polypeptides can beattached at respective addresses of a polypeptide array, for example,via conjugation to a nucleic acid (e.g., a nucleic acid nanostructure,SNAP, a complex thereof, or a component thereof) at the addresses. Thesignal change can optionally be detected at one or more address on anarray.

In a first configuration of the above method, one or more types of aminoacids in the polypeptide can be attached to a label that uniquelyidentifies the type of amino acid. In this configuration, the change insignal that identifies the amino acid can be loss of signal from therespective label. Exemplary compositions and techniques that can be usedto remove amino acids from a polypeptide and detect signal changes areset forth in Swaminathan et al., Nature Biotech. 36:1076-1082 (2018); orU.S. Pat. Nos. 9,625,469 or 10,545,153, each of which is incorporatedherein by reference. The polypeptide can be attached to a solid supportvia conjugation to a SNAP or SNAP complex.

In a second configuration of the above method, the terminal amino acidof the polypeptide can be recognized by a binding reagent that isspecific for the terminal amino acid or specific for a label moiety thatis present on the terminal amino acid. The binding reagent can bedetected on the array, for example, due to a label on the bindingreagent. Exemplary binding reagents and detection methods are set forthin US Pat. App. Pub. No. 2019/0145982 A1; 2020/0348308 A1; or2020/0348307 A1, each of which is incorporated herein by reference. Thepolypeptide can be attached to a solid support via conjugation to anucleic acid (e.g., a nucleic acid nanostructure, SNAP, a complexthereof, or a component thereof).

A method of detecting a polypeptide can include a process of detecting asample polypeptide of an array of polypeptides, the process includingsteps of (i) exposing a terminal amino acid on a polypeptide at anaddress of an array; (ii) binding a binding reagent to the terminalamino acid, where the binding reagent comprises a nucleic acid tag, andwhere a primer nucleic acid is present at the address; (iii) extendingthe primer nucleic acid, thereby producing an extended primer having acopy of the tag; and (iv) detecting the tag of the extended primer. Theterminal amino acid can be exposed, for example, by removal of one ormore amino acids from the amino terminus or carboxyl terminus of thepolypeptide. Steps (i) through (iv) can be repeated to produce a seriesof tags that is indicative of the sequence for the polypeptide. Theextending of the primer can be carried out, for example, bypolymerase-based extension of the primer, using the nucleic acid tag asa template. Alternatively, the extending of the primer can be carriedout, for example, by ligase- or chemical-based ligation of the primer toa nucleic acid that is hybridized to the nucleic acid tag. The nucleicacid tag can be detected via hybridization to nucleic acid probes (e.g.,in a microarray), amplification-based detections (e.g. PCR-baseddetection, or rolling circle amplification-based detection) or nucleiacid sequencing (e.g. cyclical reversible terminator methods, nanoporemethods, or single molecule, real time detection methods). Exemplarymethods that can be used for detecting polypeptides using nucleic acidtags are set forth in US Pat. App. Pub. No. 2019/0145982 A1;2020/0348308 A1; or 2020/0348307 A1, each of which is incorporatedherein by reference. A polypeptide, primer nucleic acid or templatenucleic acid copied by extension of the primer can be attached to a SNAPor SNAP complex.

A method of detecting can include determining a detected property suchas a polypeptide sequence, presence of a known epitope, polypeptidesize, polypeptide isoelectric point, polypeptide hydrophobicity,polypeptide hydrodynamic radius, polypeptide pKa, the presence of apost-translational modification, the absence of a post-translationalmodification, polypeptide charge, the presence of a non-natural aminoacid or other non-natural amino acid chemical unit, the presence ofsecondary, tertiary, or quaternary structure, the absence of secondary,tertiary, or quaternary structure, presence of a bound molecule, orabsence of a bound molecule. A bound non-polypeptide molecule maycomprise a chelated ion, a bound metal cluster, a bound cofactor (e.g.,a porphyrin), a bound ligand, a bound substrate, or a bound biomolecule(e.g., polysaccharide, nucleic acid, protein, etc.).

A method or apparatus of the present disclosure can optionally beconfigured for optical detection (e.g., luminescence detection).Analytes or other entities can be detected, and optionally distinguishedfrom each other, based on measurable characteristics such as thewavelength of radiation that excites a luminophore, the wavelength ofradiation emitted by a luminophore, the intensity of radiation emittedby a luminophore (e.g., at particular detection wavelength(s)),luminescence lifetime (e.g. the time that a luminophore remains in anexcited state) or luminescence polarity. Other optical characteristicsthat can be detected, and optionally used to distinguish analytes,include, for example, absorbance of radiation, resonance Raman,radiation scattering, or the like. A luminophore can be an intrinsicmoiety of a protein or other analyte to be detected, or the luminophorecan be an exogenous moiety that has been synthetically added to aprotein or other analyte.

A method or apparatus of the present disclosure can use a light sensingdevice that is appropriate for detecting a characteristic set forthherein or known in the art. Particularly useful components of a lightsensing device can include, but are not limited to, optical sub-systemsor components used in nucleic acid sequencing systems. Examples ofuseful sub systems and components thereof are set forth in US Pat. App.Pub. No. 2010/0111768 A1 or U.S. Pat. Nos. 7,329,860; 8,951,781 or9,193,996, each of which is incorporated herein by reference. Otheruseful light sensing devices and components thereof are described inU.S. Pat. Nos. 5,888,737; 6,175,002; 5,695,934; 6,140,489; or 5,863,722;or US Pat. Pub. Nos. 2007/007991 A1, 2009/0247414 A1, or 2010/0111768;or WO2007/123744, each of which is incorporated herein by reference.Light sensing devices and components that can be used to detectluminophores based on luminescence lifetime are described, for example,in U.S. Pat. Nos. 9,678,012; 9,921,157; 10,605,730; 10,712,274;10,775,305; or 10,895,534, each of which is incorporated herein byreference.

Luminescence lifetime can be detected using an integrated circuit havinga photodetection region configured to receive incident photons andproduce a plurality of charge carriers in response to the incidentphotons. The integrated circuit can include at least one charge carrierstorage region and a charge carrier segregation structure configured toselectively direct charge carriers of the plurality of charge carriersdirectly into the charge carrier storage region based upon times atwhich the charge carriers are produced. See, for example, U.S. Pat. Nos.9,606,058, 10,775,305, and 10,845,308, each of which is incorporatedherein by reference. Optical sources that produce short optical pulsescan be used for luminescence lifetime measurements. For example, a lightsource, such as a semiconductor laser or LED, can be driven with abipolar waveform to generate optical pulses with FWHM durations as shortas approximately 85 picoseconds having suppressed tail emission. See,for example, in U.S. Pat. No. 10,605,730, which is incorporated hereinby reference.

For configurations that use optical detection (e.g., luminescentdetection), one or more analytes (e.g. proteins) may be immobilized on asurface, and this surface may be scanned with a microscope to detect anysignal from the immobilized analytes. The microscope itself may comprisea digital camera or other luminescence detector configured to record,store, and analyze the data collected during the scan. A luminescencedetector of the present disclosure can be configured for epiluminescentdetection, total internal reflection (TIR) detection, waveguide assistedexcitation, or the like.

A light sensing device may be based upon any suitable technology, andmay be, for example, a charged coupled device (CCD) sensor thatgenerates pixelated image data based upon photons impacting locations inthe device. It will be understood that any of a variety of other lightsensing devices may also be used including, but not limited to, adetector array configured for time delay integration (TDI) operation, acomplementary metal oxide semiconductor (CMOS) detector, an avalanchephotodiode (APD) detector, a Geiger-mode photon counter, aphotomultiplier tube (PMT), charge injection device (CID) sensors, JOTimage sensor (Quanta), or any other suitable detector. Light sensingdevices can optionally be coupled with one or more excitation sources,for example, lasers, light emitting diodes (LEDs), arc lamps or otherenergy sources known in the art.

An optical detection system can be configured for single moleculedetection. For example, waveguides or optical confinements can be usedto deliver excitation radiation to locations of a solid support whereanalytes are located. Zero-mode waveguides can be particularly useful,examples of which are set forth in U.S. Pat. Nos. 7,181,122, 7,302,146,or 7,313,308, each of which is incorporated herein by reference.Analytes can be confined to surface features, for example, to facilitatesingle molecule resolution. For example, analytes can be distributedinto wells having nanometer dimensions such as those set forth in U.S.Pat. Nos. 7,122,482 or 8,765,359, or US Pat. App. Pub. No 2013/0116153A1, each of which is incorporated herein by reference. The wells can beconfigured for selective excitation, for example, as set forth in U.S.Pat. Nos. 8,798,414 or 9,347,829, each of which is incorporated hereinby reference. Analytes can be distributed to nanometer-scale posts, suchas high aspect ratio posts which can optionally be dielectric pillarsthat extend through a metallic layer to improve detection of an analyteattached to the pillar. See, for example, U.S. Pat. Nos. 8,148,264,9,410,887 or 9,987,609, each of which is incorporated herein byreference. Further examples of nanostructures that can be used to detectanalytes are those that change state in response to the concentration ofanalytes such that the analytes can be quantitated as set forth in WO2020/176793 A1, which is incorporated herein by reference.

An apparatus or method set forth herein need not be configured foroptical detection. For example, an electronic detector can be used fordetection of protons or charged labels (see, for example, US Pat. App.Pub. Nos. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or2010/0282617 A1, each of which is incorporated herein by reference inits entirety). A field effect transistor (FET) can be used to detectanalytes or other entities, for example, based on proximity of a fielddisrupting moiety to the FET. The field disrupting moiety can be due toan extrinsic label attached to an analyte or affinity agent, or themoiety can be intrinsic to the analyte or affinity agent being used.Surface plasmon resonance can be used to detect binding of analytes oraffinity agents at or near a surface. Exemplary sensors and methods forattaching molecules to sensors are set forth in US Pat. App. Pub. Nos.2017/0240962 A1; 2018/0051316 A1; 2018/0112265 A1; 2018/0155773 A1 or2018/0305727 A1; or U.S. Pat. Nos. 9,164,053; 9,829,456; 10,036,064,each of which is incorporated herein by reference.

A composition, apparatus or method of the present disclosure can be usedto characterize or identify at least about 0.0000001%, 0.000001%,0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 25%, 50%, 90%, 99%,99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, 99.999999%, or more of allprotein species in a proteome. Alternatively or additionally, aproteomic characterization method may characterize or no more than about99.999999%, 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99%, 90%, 50%,25%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%,0.0000001%, or less of all protein species in a proteome.

In some configurations of the compositions, apparatus and methods setforth herein, one or more proteins can be present on a solid support,where the proteins can optionally be detected. For example, a proteincan be attached to a solid support, the solid support can be contactedwith a detection agent (e.g., affinity agent) in solution, the affinityagent can interact with the protein, thereby producing a detectablesignal, and then the signal can be detected to determine the presence,absence, quantity, a characteristic or identity of the protein. Inmultiplexed versions of this approach, different proteins can beattached to different addresses in an array, and the detection steps canoccur in parallel, such that proteins at each address are detected,quantified, characterized, or identified. In another example, detectionagents can be attached to a solid support, the support can be contactedwith proteins in solution, the proteins can interact with the detectionagents, thereby producing a detectable signal, and then the signal canbe detected to determine the presence of the proteins. This approach canalso be multiplexed by attaching different probes to different addressesof an array.

In multiplexed configurations, different proteins can be attached todifferent unique identifiers (e.g. addresses in an array), and theproteins can be manipulated and detected in parallel. For example, afluid containing one or more different affinity agents can be deliveredto an array such that the proteins of the array are in simultaneouscontact with the affinity agent(s). Moreover, a plurality of addressescan be observed in parallel allowing for rapid detection of bindingevents. A plurality of different proteins can have a complexity of atleast 5, 10, 100, 1×10³, 1×10⁴, 1×10⁵ or more different native-lengthprotein primary sequences. Alternatively or additionally, a proteome,proteome subfraction or other protein sample that is analyzed in amethod set forth herein can have a complexity that is at most 1×10⁵,1×10⁴, 1×10³, 100, 10, 5 or fewer different native-length proteinprimary sequences. The total number of proteins of a sample that isdetected, characterized, or identified can differ from the number ofdifferent primary sequences in the sample, for example, due to thepresence of multiple copies of at least some protein species. Moreover,the total number of proteins of a sample that is detected,characterized, or identified can differ from the number of candidateproteins suspected of being in the sample, for example, due to thepresence of multiple copies of at least some protein species, absence ofsome proteins in a source for the sample, or loss of some proteins priorto analysis.

A particularly useful multiplex format uses an array in which proteinsand/or affinity agents are attached to unique identifiers such asaddresses on a surface. A protein can be attached to a unique identifierusing any of a variety of means. The attachment can be covalent ornon-covalent. Exemplary covalent attachments include chemical linkerssuch as those achieved using click chemistry or other linkages known inthe art or described in U.S. patent application Ser. No. 17/062,405,which is incorporated herein by reference. Non-covalent attachment canbe mediated by receptor-ligand interactions (e.g. (strept)avidin-biotin,antibody-antigen, or complementary nucleic acid strands), for example,wherein the receptor is attached to the unique identifier and the ligandis attached to the protein or vice versa. In particular configurations,a protein is attached to a solid support (e.g., an address in an array)via a structured nucleic acid particle (SNAP). A protein can be attachedto a SNAP and the SNAP can interact with a solid support, for example,by non-covalent interactions of the DNA with the support and/or viacovalent linkage of the SNAP to the support. Nucleic acid origami ornucleic acid nanoballs are particularly useful. The use of SNAPs andother moieties to attach proteins to unique identifiers such as tags oraddresses in an array are set forth in U.S. patent application Ser. No.17/062,405, which is incorporated herein by reference.

The methods, compositions and apparatus of the present disclosure areparticularly well suited for use with proteins. Although proteins areexemplified throughout the present disclosure, it will be understoodthat other analytes can be similarly used. Exemplary analytes include,but are not limited to, biomolecules, polysaccharides, nucleic acids,lipids, metabolites, hormones, vitamins, enzyme cofactors, therapeuticagents, candidate therapeutic agents or combinations thereof. An analytecan be a non-biological atom or molecule, such as a synthetic polymer,metal, metal oxide, ceramic, semiconductor, mineral, or a combinationthereof.

One or more proteins that are used in a method, composition or apparatusherein, can be derived from a natural or synthetic source. Exemplarysources include, but are not limited to biological tissues, fluids,cells or subcellular compartments (e.g., organelles). For example, asample can be derived from a tissue biopsy, biological fluid (e.g.,blood, sweat, tears, plasma, extracellular fluid, urine, mucus, saliva,semen, vaginal fluid, synovial fluid, lymph, cerebrospinal fluid,peritoneal fluid, pleural fluid, amniotic fluid, intracellular fluid,extracellular fluid, etc.), fecal sample, hair sample, cultured cell,culture media, fixed tissue sample (e.g., fresh frozen or formalin-fixedparaffin-embedded) or product of a protein synthesis reaction. A proteinsource may include any sample where a protein is a native or expectedconstituent. For example, a primary source for a cancer biomarkerprotein may be a tumor biopsy sample or bodily fluid. Other sourcesinclude environmental samples or forensic samples.

Exemplary organisms from which proteins or other analytes can be derivedinclude, for example, a mammal such as a rodent, mouse, rat, rabbit,guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate,non-human primate or human; a plant such as Arabidopsis thaliana,tobacco, corn, sorghum, oat, wheat, rice, canola, or soybean; an algaesuch as Chlamydomonas reinhardtii; a nematode such as Caenorhabditiselegans; an insect such as Drosophila melanogaster, mosquito, fruit fly,honey bee or spider; a fish such as zebrafish; a reptile; an amphibiansuch as a frog or Xenopus laevis; a Dictyostelium discoideum; a fungisuch as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a Plasmodium falciparum.Proteins can also be derived from a prokaryote such as a bacterium,Escherichia coli, staphylococci or Mycoplasma pneumoniae; an archae; avirus such as Hepatitis C virus, influenza virus, coronavirus, or humanimmunodeficiency virus; or a viroid. Proteins can be derived from ahomogeneous culture or population of the above organisms oralternatively from a collection of several different organisms, forexample, in a community or ecosystem.

In some cases, a protein or other biomolecule can be derived from anorganism that is collected from a host organism. For example, a proteinmay be derived from a parasitic, pathogenic, symbiotic, or latentorganism collected from a host organism. A protein can be derived froman organism, tissue, cell or biological fluid that is known or suspectedof being linked with a disease state or disorder (e.g., cancer).Alternatively, a protein can be derived from an organism, tissue, cellor biological fluid that is known or suspected of not being linked to aparticular disease state or disorder. For example, the proteins isolatedfrom such a source can be used as a control for comparison to resultsacquired from a source that is known or suspected of being linked to theparticular disease state or disorder. A sample may include a microbiomeor substantial portion of a microbiome. In some cases, one or moreproteins used in a method, composition or apparatus set forth herein maybe obtained from a single source and no more than the single source. Thesingle source can be, for example, a single organism (e.g. an individualhuman), single tissue, single cell, single organelle (e.g. endoplasmicreticulum, Golgi apparatus or nucleus), or single protein-containingparticle (e.g., a viral particle or vesicle).

A method, composition or apparatus of the present disclosure can use orinclude a plurality of proteins having any of a variety of compositionssuch as a plurality of proteins composed of a proteome or fractionthereof. For example, a plurality of proteins can include solution-phaseproteins, such as proteins in a biological sample or fraction thereof,or a plurality of proteins can include proteins that are immobilized,such as proteins attached to a particle or solid support. By way offurther example, a plurality of proteins can include proteins that aredetected, analyzed, or identified in connection with a method,composition or apparatus of the present disclosure. The content of aplurality of proteins can be understood according to any of a variety ofcharacteristics such as those set forth below or elsewhere herein.

A plurality of proteins can be characterized in terms of total proteinmass. The total mass of protein in a liter of plasma has been estimatedto be 70 grams and the total mass of protein in a human cell has beenestimated to be between 100 picograms (pg) and 500 pg depending uponcells type. See Wisniewski et al. Molecular & Cellular Proteomics13:10.1074/mcp.M113.037309, 3497-3506 (2014), which is incorporatedherein by reference. A plurality of proteins used or included in amethod, composition or apparatus set forth herein can include at least 1pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 10mg, 100 mg or more protein by mass. Alternatively or additionally, aplurality of proteins may contain at most 100 mg, 10 mg, 1 mg, 100 μg,10 μg, 1 μg, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg or less protein bymass.

A plurality of proteins can be characterized in terms of percent massrelative to a given source such as a biological source (e.g. cell,tissue, or biological fluid such as blood). For example, a plurality ofproteins may contain at least 60%, 75%, 90%, 95%, 99%, 99.9% or more ofthe total protein mass present in the source from which the plurality ofproteins was derived. Alternatively or additionally, a plurality ofproteins may contain at most 99.9%, 99%, 95%, 90%, 75%, 60% or less ofthe total protein mass present in the source from which the plurality ofproteins was derived.

A plurality of proteins can be characterized in terms of total number ofprotein molecules. The total number of protein molecules in aSaccharomyces cerevisiae cell has been estimated to be about 42 millionprotein molecules. See Ho et al., Cell Systems (2018), DOI:10.1016/j.cels.2017.12.004, which is incorporated herein by reference. Aplurality of proteins used or included in a method, composition orapparatus set forth herein can include at least 1 protein molecule, 10protein molecules, 100 protein molecules, 1×10⁴ protein molecules, 1×10⁶protein molecules, 1×10⁸ protein molecules, 1×10¹⁰ protein molecules, 1mole (6.02214076×10²³ molecules) of protein, 10 moles of proteinmolecules, 100 moles of protein molecules or more. Alternatively oradditionally, a plurality of proteins may contain at most 100 moles ofprotein molecules, 10 moles of protein molecules, 1 mole of proteinmolecules, 1×10¹⁰ protein molecules, 1×10⁸ protein molecules, 1×10⁶protein molecules, 1×10⁴ protein molecules, 100 protein molecules, 10protein molecules, 1 protein molecule or less.

A plurality of proteins can be characterized in terms of the variety offull-length primary protein structures in the plurality. For example,the variety of full-length primary protein structures in a plurality ofproteins can be equated with the number of different protein-encodinggenes in the source for the plurality of proteins. Whether or not theproteins are derived from a known genome or from any genome at all, thevariety of full-length primary protein structures can be countedindependent of presence or absence of post translational modificationsin the proteins. A human proteome is estimated to have about 20,000different protein-encoding genes such that a plurality of proteinsderived from a human can include up to about 20,000 different primaryprotein structures. See Aebersold et al., Nat. Chem. Biol. 14:206-214(2018), which is incorporated herein by reference. Other genomes andproteomes in nature are known to be larger or smaller. A plurality ofproteins used or included in a method, composition or apparatus setforth herein can have a complexity of at least 2, 5, 10, 100, 1×10³,1×10⁴, 2×10⁴, 3×10⁴ or more different full-length primary proteinstructures. Alternatively or additionally, a plurality of proteins canhave a complexity that is at most 3×10⁴, 2×10⁴, 1×10⁴, 1×10³, 100, 10,5, 2 or fewer different full-length primary protein structures.

In relative terms, a plurality of proteins used or included in a method,composition or apparatus set forth herein may contain at least onerepresentative for at least 60%, 75%, 90%, 95%, 99%, 99.9% or more ofthe proteins encoded by the genome of a source from which the sample wasderived. Alternatively or additionally, a plurality of proteins maycontain a representative for at most 99.9%, 99%, 95%, 90%, 75%, 60% orless of the proteins encoded by the genome of a source from which thesample was derived.

A plurality of proteins can be characterized in terms of the variety ofprimary protein structures in the plurality including transcribed splicevariants. The human proteome has been estimated to include about 70,000different primary protein structures when splice variants are included.See Aebersold et al., Nat. Chem. Biol. 14:206-214 (2018), which isincorporated herein by reference. Moreover, the number of thepartial-length primary protein structures can increase due tofragmentation that occurs in a sample. A plurality of proteins used orincluded in a method, composition or apparatus set forth herein can havea complexity of at least 2, 5, 10, 100, 1×10³, 1×10⁴, 7×10⁴, 1×10⁵,1×10⁶ or more different primary protein structures. Alternatively oradditionally, a plurality of proteins can have a complexity that is atmost 1×10⁶, 1×10⁵, 7×10⁴, 1×10⁴, 1×10³, 100, 10, 5, 2 or fewer differentprimary protein structures.

A plurality of proteins can be characterized in terms of the variety ofprotein structures in the plurality including different primarystructures and different proteoforms among the primary structures.Different molecular forms of proteins expressed from a given gene areconsidered to be different proteoforms. Protoeforms can differ, forexample, due to differences in primary structure (e.g., shorter orlonger amino acid sequences), different arrangement of domains (e.g.transcriptional splice variants), or different post translationalmodifications (e.g. presence or absence of phosphoryl, glycosyl, acetyl,or ubiquitin moieties). The human proteome is estimated to includehundreds of thousands of proteins when counting the different primarystructures and proteoforms. See Aebersold et al., Nat. Chem. Biol.14:206-214 (2018), which is incorporated herein by reference. Aplurality of proteins used or included in a method, composition orapparatus set forth herein can have a complexity of at least 2, 5, 10,100, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 5×10⁶, 1×10⁷ or more different proteinstructures. Alternatively or additionally, a plurality of proteins canhave a complexity that is at most 1×10⁷, 5×10⁶, 1×10⁶, 1×10⁵, 1×10⁴,1×10³, 100, 10, 5, 2 or fewer different protein structures.

A plurality of proteins can be characterized in terms of the dynamicrange for the different protein structures in the sample. The dynamicrange can be a measure of the range of abundance for all differentprotein structures in a plurality of proteins, the range of abundancefor all different primary protein structures in a plurality of proteins,the range of abundance for all different full-length primary proteinstructures in a plurality of proteins, the range of abundance for alldifferent full-length gene products in a plurality of proteins, therange of abundance for all different proteoforms expressed from a givengene, or the range of abundance for any other set of different proteinsset forth herein. The dynamic range for all proteins in human plasma isestimated to span more than 10 orders of magnitude from albumin, themost abundant protein, to the rarest proteins that have been measuredclinically. See Anderson and Anderson Mol Cell Proteomics 1:845-67(2002), which is incorporated herein by reference. The dynamic range forplurality of proteins set forth herein can be a factor of at least 10,100, 1×10³, 1×10⁴, 1×10⁶, 1×10⁸, 1×10¹⁰, or more. Alternatively oradditionally, the dynamic range for plurality of proteins set forthherein can be a factor of at most 1×10¹⁰, 1×10⁸, 1×10⁶, 1×10⁴, 1×10³,100, 10 or less.

EXAMPLES Example 1 Conjugation of Proteins to SNAPs

MTz-functionalized proteins are conjugated to TCO-functionalized DNAorigami SNAP complexes comprising one or more TCO functional groups.Each TCO-functionalized DNA origami SNAP complex comprises a tile-shapeddisplay SNAP comprising a TCO-functionalized polypeptide binding groupthat is coupled to four tile-shaped utility SNAPs. Each display SNAPcomprises either 1 or 4 TCO binding groups. The TCO-functionalized DNAorigami is provided in a buffer comprising 200 mM NaCl, 5 mM Tris-HCl,11 mM MgCl₂, and 1 mM EDTA at pH 8.0. The amount of mTz-modified proteinis calculated based upon the amount of tile to be used in theconjugation reaction. The volume of protein added to the conjugationreaction is calculated according to equation (1):y=(xC _(x) wz)/C _(y)  (1)

-   -   Where y=total volume of mTz-functionalized protein (μl)        -   x=total volume of DNA origami (μl)        -   C_(x)=concentration of DNA origami (μM)        -   C_(y)=concentration of mTz-functionalized protein (μM)        -   w=molar equivalents of protein to TCO        -   z=number of TCO moieties per DNA origami molecule

Volumes of mTz-functionalized protein and TCO-DNA origami are combinedaccording to the amounts calculated in equation (1). If the volume ofmTz-functionalized protein in the reaction mixture exceeds 10% of thetotal volume (x+y), additional MgCl₂ must be added to maintain themagnesium concentration of the reaction mixture. If necessary, 1 μl ofMgCl₂ should be added to the protein prior to the addition of the DNAorigami at a concentration according to equation (2):C _(M)=12.4y+12.4  (2)

-   -   Where C_(M)=concentration of MgCl₂ (mM)

The reaction mixture is gently mixed, then placed on a thermomixer orthermocycler at 25° C. The reaction tube is jacketed to prevent exposureto light. Reactions with a 10-fold or higher excess of protein areincubated for 5 hours or more. Reactions with less than a 10-fold excessof protein are incubated for 16 hours or more to ensure completereaction of mTz with TCO.

Protein conjugates are purified on an Agilent 1100 HPLC with an AgilentBio-SEC5 4.6×300 mm column. The HPLC solvent is filtered 200 mM NaCl, 5mM Tris-HCl, 11 mM MgCl₂, and 1 mM EDTA at pH 8.0. The HPLC is run withisocratic flow at 0.3 ml/min for 25 minutes. Fractions are collected in30 s intervals between 5 min and 13 mins of the run. Detection ofDNA-containing fractions is performed at 260 nm wavelength, withDNA-containing fractions pooled. Pooled DNA-containing fractions areconcentrated to a total volume of about 100 μl.

Example 2 Analysis of Protein Conjugates

Protein conjugates of Protein A, maltose-binding protein (MBP), andubiquitin were formed by a mTz-TCO conjugation chemistry. Proteinconjugates were formed with DNA origami containing a single TCO moiety.Single-TCO DNA origami were conjugated to fluorescently-labeled versionof the three aforementioned proteins. Protein A was labeled with anAlexa-Fluor 647 fluorescent dye. MBP was labeled with an Alexa-Fluor 488fluorescent dye. Ubiquitin was labeled with tetramethylrhodamine (˜555nm wavelength). A control reaction was run using mTz-functionalizedprotein with DNA origami containing no TCO moiety.

Fluorescently-labeled protein conjugates were run on an Agilent 1100HPLC with an Agilent Bio-SEC5 4.6×300 mm column. The HPLC solvent wasfiltered 200 mM NaCl, 5 mM Tris-HCl, 11 mM MgCl₂, and 1 mM EDTA at pH8.0. The HPLC was run with isocratic flow at 0.3 ml/min for 25 minutes.The HPLC monitored light absorption across a range of wavelengthsbetween 190 nm and 800 nm. 260 nm wavelength was used to determine thepresence of DNA. 488 nm, 553 nm, and 652 nm wavelengths were used todetermine the presence of fluorescently-labeled protein as appropriate.

FIG. 30A shows HPLC data for Protein A conjugates. The upperchromatogram depicts 260 nm data, showing the elution of DNA origamiaround 11 mins. The lower chromatogram depicts 652 nm data, showingelution of protein around 11 mins, with excess unconjugated proteinfollowing at around 15 mins. Negative control data shown in FIG. 30Bshows no protein eluting with the DNA origami at 11 mins (lowerchromatogram) due to available TCO to complete the conjugation.

FIG. 30C shows HPLC data for MBP protein conjugates. The lowerchromatogram depicts 260 nm data, showing the elution of DNA origamiaround 11 mins. The upper chromatogram depicts 488 nm data, showingelution of protein around 11 mins, with excess unconjugated proteinfollowing at around 15 mins. FIG. 30D shows HPLC data for ubiquitinprotein conjugates. The upper chromatogram depicts 260 nm data, showingthe elution of DNA origami around 11 mins. The lower chromatogramdepicts 553 nm data, showing elution of protein around 11 mins, withexcess unconjugated protein following at around 15 mins.

Example 3 Deposition of SNAPs

Anchoring groups comprising 5-tile DNA origami are deposited on a glasssubstrate. A schematic of the basic structure of 5-tile origami is shownin FIG. 31 . The origami complexes comprise four edge tiles 3110 thatare joined to a central tile 3120 at a hybridization region 3140. Thecentral tile 3120 comprises a reactive handle 3130 that is configured toconjugate a functionalized protein. DNA origami are labeled withAlexa-Fluor 488 dye to make them optically detectable. The glasssubstrate is a Nexterion D263 170 μm-thick glass slide that has beencoated with a uniform monolayer of (3-aminopropyl)trimethoxysilane(APTMS).

Prior to deposition of the anchoring groups, the glass substrate isincubated in a deposition buffer solution containing 5 mM Tris-HCl—pH8.0, 205 mM NaCl, 1 mM EDTA, and 12.5 mM MgCl₂ for 1 hour. 10 μl of5-tile DNA origami at 2 ng/μl (91 pM) is applied to the glass substratein a deposition buffer containing 5 mM Tris-HCl—pH 8.0, 205 mM NaCl, 1mM EDTA, and 12.5 mM MgCl₂. The DNA origami are applied to the glasssubstrate slowly to prevent shearing. The DNA origami are incubated onthe substrate for 10 minutes. After incubation, excess DNA origami areremoved from the substrate by a 0.5 ml wash with a buffer containing 1×Neoventures buffer (10 mM HEPES, 120 mM NaCl, 5 mM MgCl₂, and 5 mM KCl,pH 7.4), 0.1% Tween-20, and 0.001% Lipidure CM5206. Additional MgCl₂ isadded to the wash buffer to bring the total MgCl₂ concentration to 10mM. Deposited DNA origami may be imaged by excitation of the labeled DNAorigami with 488 nm light.

Example 4 SNAP Deposition Conditions

Anchoring group deposition was studied under differing depositionsolvents. 5-tile DNA origami were deposited on a glass substrate. Thedeposition buffers utilized were: 1) DNA origami buffer (5 mMTris-HCl—pH 8.0, 205 mM NaCl, 1 mM EDTA, and 12.5 mM MgCl₂); 2) DNAorigami buffer with an additional 2.5 M NaCl added; and 3) DNA origamibuffer with 0.01% Tween-20. DNA origami were deposited on the glasssubstrate according to the method described in Example 3. Each bufferwas utilized for the pre-deposition incubation and the deposition step.Control substrates were prepared by cleaning Nexterion D263 170 μm-thickglass slide with O₂ plasma (no APTMS coating), then following thedeposition method of Example 3. Each Nexterion D263 glass slide wasjoined to a second glass slide with an inward-facing PEG 3-6 surfacecoating to form a 3-lane flow cell with a deposition area on the glasssubstrate of each lane. Each lane of each flow cell corresponded to oneof the three tested deposition buffers. Deposition on APTMS-coatedsubstrate was tested for 3 different flow cells. Deposition on theuncoated substrate was tested for 3 different flow cells.

All glass substrates were imaged at 30 locations by confocal scanninglaser microscopy at 488 nm. Pixel intensity counts were performed foreach image by an image analysis software. Pixel intensity counts acrossthe series of 30 images for each slide were averaged to provide averagefluorescence intensity.

FIGS. 32A and 32B show confocal scanning image results for DNA origamideposition under DNA origami buffer for the APTMS-coated substrate (FIG.32A) and the uncoated substrate (FIG. 32B). Individual DNA origami canbe seen at discrete locations on the surface of the coated substrate.Minimal deposition is apparent on the uncoated substrate. FIGS. 32C and32D show confocal scanning image results for DNA origami depositionunder DNA origami buffer with 2.5 M NaCl for the APTMS-coated substrate(FIG. 32C) and the uncoated substrate (FIG. 32D). Individual DNA origamican be seen at discrete locations on the surface of the coatedsubstrate, although less deposition appears to occur compared to DNAorigami buffer without 2.5 M NaCl. Minimal deposition is apparent on theuncoated substrate. FIGS. 32E and 32F show confocal scanning imageresults for DNA origami deposition under DNA origami buffer with 0.01%Tween-20 for the APTMS-coated substrate (FIG. 32E) and the uncoatedsubstrate (FIG. 32F). Individual DNA origami can be seen at discretelocations on the surface of the coated substrate. No deposition isapparent on the uncoated substrate. FIG. 33 shows average totalanchoring group counts for images collected under each buffer for eachtested flow cell. The left data series shows results for theAPTMS-coated substrate. The right data series shows results for theuncoated substrate. DNA origami are shown to deposit on the coatedsubstrate with standard DNA origami buffer, or in the presence of highsalt concentration or surfactants. Minimal deposition of DNA origami isobserved on the uncoated substrate. The differences in total depositionon the substrate between different buffer compositions suggests thatsolvent composition can affect the quantity and density of anchoringgroups on the substrate surface.

Example 5 Deposition of Protein Conjugates

Protein conjugates were deposited on glass substrate coated with a layerof APTMS according to the method described in Example 4. The proteinconjugates comprised a 5-tile DNA origami conjugated to maltose bindingprotein (MBP) via a covalent methyltetrazine-transcyclooctene linkage.MBP protein conjugates were labeled with Alexa-Fluor 647 fluorophores topermit detection of protein conjugate deposition. Deposition of each MBPprotein conjugate was observed in the same buffering conditionsdescribed in Example 4 (DNA origami buffer with or without 2.5 M NaCl or0.01% Tween-20). Deposition of MBP protein conjugates under DNA origamibuffer was tested in two separate flow cells. Deposition of MBP proteinconjugates in the presence of 2.5 M NaCl or 0.01% Tween-20 was tested inthree separate flow cells. Flows cells incubated with buffers containingno protein conjugates were also observed as negative controls.

FIGS. 34A-34C show confocal scanning image results for DNA origamideposition under different DNA origami buffer compositions for theAPTMS-coated substrate. FIG. 34A shows individual MBP protein conjugatesthat were deposited in DNA origami buffer. FIG. 34B shows individual MBPprotein conjugates that were deposited in DNA origami buffer containing2.5 M NaCl. FIG. 34C shows individual MBP protein conjugates that weredeposited in DNA origami buffer containing 0.01% Tween-20. IndividualDNA origami can be seen at discrete locations on the surface of eachAPTMS coated substrate. FIG. 35 shows average total protein conjugatecounts collected under each buffer for each tested flow cell. Dataalternates between flow cells tested with protein conjugates and flowcells tested without protein conjugates. The leftmost four counts werefor DNA origami buffer only. The middle six counts were for DNA origamibuffer containing 2.5 M NaCl. The rightmost six counts were for DNAorigami buffer containing 0.01% Tween-20. Deposition of proteinconjugates on the APTMS-coated glass substrate was observed for allsubstrates, with slightly lower counts observed in the presence of 2.5 MNaCl, and slightly higher counts observed in the presence of 0.01%Tween-20. Anchoring groups are observed to efficiently deposit on anAPTMS-coated substrate after the formation of protein conjugates.

Example 6 Deposition of Protein Conjugates

Protein conjugates were deposited on a patterned Nexterion D263 glasschip comprising square pattern of binding sites. The patterned region ofeach glass chip contained a polypeptide binding region having over 190million binding sites. The polypeptide binding region was patterned with12544 subgrids, with each subgrid containing 123×123 binding sites in asquare configuration (15129 total binding sites per subgrid). Glass chipsurfaces were coated with a layer of APTMS. The protein conjugatescomprised a 5-tile DNA origami conjugated to his-tagged ubiquitin(Ubi-His) via a covalent methyltetrazine-transcyclooctene linkage. TheDNA origami of the Ubi-His protein conjugates were labeled withAlexa-Fluor 488 fluorophores to permit detection of protein conjugatedeposition. 15 μl of 0.3 nM protein conjugates were incubated on thechip for 10 minutes in a DNA origami buffer, then rinsed with 40 μl of arinsing buffer containing 200 mM HEPES, 2.4 M NaCl, 100 mM MgCl₂, 100 mMKCl, 0.1% Tween-20, and 0.001% Lipidure CM5206 at pH 7.4. After rinsing,glass chips were imaged by confocal laser scanning microscopy at 488 nmto detect deposited protein conjugates on the patterned glass surface.After the initial imaging, chips were incubated with a blocking buffercontaining the same components as the rinsing buffer with 100 mg/mldextran sulfate. Chips were incubated with 40 μl of blocking buffer for60 mins, then rinsed again with 40 μl of rinsing buffer. Chips weresubsequently incubated with 25 μl of B1 aptamer (his-tag affinitytarget) labeled with Alexa-Fluor 647 nm fluorescent dye. Chips wereimaged at 647 nm to using a Thorlabs confocal laser scanning microscope.

FIG. 21A shows fluorescence microscopy results at 488 nm for DNAorigami-Ubi-His conjugates deposited on the patterned glass arrays. DNAorigami are observed to have deposited on the array with nearly completeoccupancy of binding sites. FIG. 21B shows imaging at 647 nm of the samedeposited Ubi-His conjugates imaged with B1 aptamer (positive control).When imaged with the his-tag specific labeled affinity reagents, thegrid deposition pattern is again observed, confirming theco-localization of the DNA origami and the conjugated proteins.

Example 7 SNAP Synthesis and Purification

A plurality of tile-shaped SNAPs are formed by combining M13 phagegenome scaffold strands with pluralities of 218 differingoligonucleotides, including a plurality of TCO-terminatedoligonucleotides that are configured to couple to an analyte. Theoligonucleotides are combined in a DNA origami buffer comprising 100 mMMgCl₂ and heated to 95° C. After heating, the oligonucleotides areallowed to slowly cool to 20° C. thereby permitting annealing ofoligonucleotides into SNAP structures. After SNAP formation, SNAPs arepurified from excess oligonucleotides on an HPLC system containing asize-exclusion chromatography column. Surprisingly, it is found that aglycan-specific column effectively purifies formed SNAPs with minimalresidual oligonucleotides or other unwanted components.

Example 8 SNAP Synthesis and Purification

SNAPs were synthesized via the method described in Example 7.Synthesized SNAPs were substantially square DNA origami structures withan approximately 83 nanometer (nm) edge length. Each square SNAPcontained 65 oligonucleotides with pendant handles for binding ofadditional components to a SNAP via complementary oligonucleotideconjugation to pendant groups: 1 pendant single-stranded DNA handle forcoupling an analyte to an upper display face, 20 pendant single-strandedDNA handles for coupling a SNAP to a surface, and 44 pendantsingle-stranded DNA handles for coupling detectable fluorescent labelsto the 4 edges of the SNAP (11 per side). All oligonucleotide sequenceswere designed using CADNANO2 software.

Table I contains sequence listings for coupling regions of SNAPoligonucleotides. SEQ. ID 1 is the sequence listing for the couplingregion of an oligonucleotide that is configured to couple to acomplementary oligonucleotide that is conjugated to an analyte. SEQ. ID2 is the sequence listing for the coupling region of an oligonucleotidethat is configured to couple to a complementary oligonucleotide that isconjugated to the surface of a solid support. SEQ. ID 3 is the sequencelisting for the coupling region of an oligonucleotide that is configuredto couple to a complementary oligonucleotide that is conjugated to afluorescent Alexa-Fluor™ 488 dye molecule.

Table II contains sequence listings for the 217 staple oligonucleotidesutilized to form the SNAPs with 20 pendant surface-linked moieties.Pendant regions of the 65 coupling oligonucleotides are highlighted inbold text. All staple oligonucleotides listed in Table III were combinedwith M13mp18 single-stranded phage genomic DNA to fold the DNA origamistructure.

TABLE II Oligo- SEQ. nucleotide ID Type 5′-3′ DNA Sequence Listing 1Analyte TTTCACTCACCTCCATCTCCACTCCTACCCA Coupling TCCAACTCCCAC 2 SurfaceTTTTACCATCTTCCTCTCCAC Coupling 3 Label  TTTAACTACTCCCACTCTCACCCTCACCCTACoupling CTCCAACTCAAC

TABLE III SEQ. ID 5′-3′ DNA Sequence Listing 4 TCATTTGCTAATAGTAGTAGCATT5 CAACTAAAGTACGGTGGGATGGCT 6 CATTATTAGCAAAAGAAGTTTTGC 7ACCCTCATTCAGGGATAGCAAGCC 8 TTAGGATTAGCGGGGTGGAACCTA 9AGGCCGGAACCAGAGCCACCACCG 10 AGAATATCAGACGACGACAATAAA 11TCATATGCGTTATACAAAGGCGTT 12 CGGGAGAATTTAATGGAAACAGTA 13GCGCGTACTTTCCTCGTTAGAATC 14 AAAGCCGGCGAACGTGTGCCGTAA 15AATTCCACGTTTGCGTATTGGGCG 16 TTAAGAGGGTCCAATACTGCGGATAGCGAG 17AGGCTTTTCAGGTAGAAAGATTCAATTACC 18 TTATGCGATTGACAAGAACCGGAGGTCAAT 19CATAAGGGACACTAAAACACTCACATTAAA 20 CGGGTAAAATTCGGTCGCTGAGGAATGACA 21GTCTCTGACACCCTCAGAGCCACATCAAAA 22 TCACCGGAAACGTCACCAATGAATTATTCA 23TTAAAGGTACATATAAAAGAAACAAACGCA 24 ATAATAACTCAGAGAGATAACCCGAAGCGC 25ATTAGACGGAGCGTCTTTCCAGAGCTACAA 26 TATATAACGTAAATCGTCGCTATATTTGAA 27TTACCTTTACAATAACGGATTCGCAAAATT 28 ATTTGCACCATTTTGCGGAACAAATTTGAG 29GATTTAGATTGCTGAACCTCAAAGTATTAA 30 CACCGCCTGAAAGCGTAAGAATACATTCTG 31TGAGTGTTCAGCTGATTGCCCTTGCGCGGG 32 GAGAGGCGACAACATACGAGCCGCTGCAGG 33TCGACTCTGAAGGGCGATCGGTGCGGCCTC 34 AGGAAGATCATTAAATGTGAGCGTTTTTAA 35CCAATAGGAAACTAGCATGTCAAGGAGCAA 36 TAGAGCTTCAGACCGGAAGCAAACCTATTATA 37GTCAGAAGATTGAATCCCCCTCAACCTCGTTT 38 AAATATTCCAAAGCGGATTGCATCGAGCTTCA 39ACCAGACGGAATACCACATTCAACGAGATGGT 40 AGATTTAGACGATAAAAACCAAAAATCGTCAT 41AGTCAGGACATAGGCTGGCTGACCTTTGAAAG 42 TTAATTTCCAACGTAACAAAGCTGTCCATGTT 43GAGTAATCTTTTAAGAACTGGCTCCGGAACAA 44 ACCCAAATAACTTTAATCATTGTGATCAGTTG 45ACTTAGCCATTATACCAAGCGCGAGAGGACTA 46 AAAAGAATAACCGAACTGACCAACTTCATCAA 47AAGACTTTGGCCGCTTTTGCGGGATTAAACAG 48 GAGTTAAATTCATGAGGAAGTTTCTCTTTGAC 49CTTGATACTGAAAATCTCCAAAAAAGCGGAGT 50 TTTCACGTCGATAGTTGCGCCGACCTTGCAGG 51TTATTCTGACTGGTAATAAGTTTTAACAAATA 52 AATCCTCAACCAGAACCACCACCAGCCCCCTT 53GAGCCGCCTTAAAGCCAGAATGGAGATGATAC 54 ATTAGCGTCCGTAATCAGTAGCGAATTGAGGG 55GCCATTTGCAAACGTAGAAAATACCTGGCATG 56 AGGGAAGGATAAGTTTATTTTGTCAGCCGAAC 57AGGTGGCAGAATTATCACCGTCACCATTAGCA 58 AAAGTTACGCCCAATAATAAGAGCAGCCTTTA 59CGCTAATAGGAATACCCAAAAGAAATACATAA 60 CAGAGAGAACAAAATAAACAGCCATTAAATCA 61AGATTAGTATATAGAAGGCTTATCCAAGCCGT 62 CAAATCAGTGCTATTTTGCACCCAGCCTAATT 63AAATAAGAACTTTTTCAAATATATCTGAGAGA 64 CTACCTTTAGAATCCTTGAAAACAAGAAAACA 65TTTCCCTTTTAACCTCCGGCTTAGCAAAGAAC 66 AAATTAATACCAAGTTACAAAATCCTGAATAA 67CTTTGAATTACATTTAACAATTTCTAATTAAT 68 GTAGATTTGTTATTAATTTTAAAAAACAATTC 69TGGAAGGGAGCGGAATTATCATCAACTAATAG 70 AACATTATGTAAAACAGAAATAAATTTTACAT 71CCAGAAGGTTAGAACCTACCATATCCTGATTG 72 ATTAGAGCAATATCTGGTCAGTTGCAGCAGAA 73GCATCACCAGTATTAGACTTTACAGTTTGAGT 74 CCTCAATCCGTCAATAGATAATACAGAAACCA 75GATAAAACTTTTTGAATGGCTATTTTCACCAG 76 AGACAATAAGAGGTGAGGCGGTCATATCAAAC 77TCACACGATGCAACAGGAAAAACGGAAGAACT 78 CCAGCCATCCAGTAATAAAAGGGACGTGGCAC 79AGCACTAAAAAGGGCGAAAAACCGAAATCCCT 80 TATAAATCGAGAGTTGCAGCAAGCGTCGTGCC 81GGCCCTGAAAAAGAATAGCCCGAGCGTGGACT 82 AGCTGCATAGCCTGGGGTGCCTAAGTAAAACG 83AAGTGTAATAATGAATCGGCCAACCACCGCCT 84 GAATTCGTGCCATTCGCCATTCAGTTCCGGCA 85ACGGCCAGTACGCCAGCTGGCGAACATCTGCC 86 ACTGTTGGAGAGGATCCCCGGGTACCGCTCAC 87TTCGCTATTGCCAAGCTTGCATGCGAAGCATA 88 AGTTTGAGATTCTCCGTGGGAACAATTCGCAT 89TTCATCAACGCACTCCAGCCAGCTGCTGCGCA 90 CCCGTCGGGGGACGACGACAGTATCGGGCCTC 91TAAATTTTTGATAATCAGAAAAGCACAAAGGC 92 ACCCCGGTTGTTAAATCAGCTCATAGTAACAA 93TATCAGGTAAATCACCATCAATATCAATGCCT 94 AGACAGTCCATTGCCTGAGAGTCTTCATATGT 95GACGGAAAACCATCGATAGCAGCATTGCCATCTTTTCATACACCCT CA 96TGCCAGTTATAACATAAAAACAGGACAAGAATTGAGTTAACAGAA GGA 97TGCCACTACTTTTTTTGCCACCCTC 98 AACTGAACATTTTTTTTGAATAACC 99GCCACGCTGTTTTTTTACCAGTGAG 100 CAAAAATAATTTTTTTTGTTTAGAC 101GATACATTTCGCTTTTTTGACCCTGTAAT 102 ACCGTACTCAGGTTTTTGATCTAAAGTTT 103AACATGTAATTTTTTTTGAAACCAATCAA 104 GCGTAACCACCATTTTTGAGTAAAAGAGT 105CAGAGGGGGTTTTGCCTTCCTGTAGCCAGCT 106 GAACCGCCTCTTTACCTAAAACGAAAGAGGC 107CATAAATCAATTTAGTCAGAGGGTAATTGAG 108 CCAGGGTGGTTTTGCAAATGAAAAATCTAAA 109ACAACCATTTTTTCATACATGGCTTTTAAGCGCA 110TTTTATCTTTTTTATCCAATCGCAAGAGTTGGGT illGCCAACATTTTTTCCACTATTAAAGAAATAGGGT 112ACAAGAGTTTTTTTCGCGTTTTAATTCAAAAAGA 113TGGATAGCAAGCCCGATTTTTAATCGTAAACGCCAT 114AGAACCGCATTTACCGTTTTACCGATATATACGTAA 115TTGCTTCTTATATGTATTTTACGCTAACGGAGAATT 116ACGGGCAAGTTCCAGTTTTTTCTGACCTGCAACAGT 117GAGAATAGAAAGGAACAACTATTTTCTCAAGAGAAGGA 118TTTTATTTTCATCGTAGGAATTTTTAGCCTGTTTAGTA 119CAAACTATCGGCCTTGCTGGTTTTTGAGCTTGACGGGG 120GAGTAATGTGTAGGTAAAGATTTTTTGTTTTAAATATG 121AGGACAGATGATTTTTTCACCAGTAGCACCATTACCGACTTGA 122ATTAAGACTCCTTTTTAATATACAGTAACAGTACCGAAATTGC 123GACAACTCGTATTTTTTCCTGTGTGAAATTGTTATCCGAGCTC 124CCGCTTCTGGTTTTTTCGTTAATAAAACGAACTAAATTATACC 125TGTCGTCTCAGCCCTCATATTTTTTTCGCCACCCTCAGGTGTATC 126TAATCGGCCATCCTAATTTTTTTTTTTTTTCGAGCCAACAACGCC 127CTGTCCATTTTTATAATCATTTTTTTCTTAATGCGCCCACGCTGC 128ACTTTTGCATCGGTTGTACTTTTTTTAACCTGTTTAGGACCATTA 129AAGCGAACAATTGCTGAATATAATGCTGTATTTTTTTGTGAGAAAG GCCGG 130AGGAGTGTAAACATGAAAGTATTAAGAGGCTTTTTTTGCGAATAAT AATTT 131GCGAGAAAATAAACACCGGAATCATAATTATTTTTTTCGCCCAATA GCAAG 132CCAACGTCATCGGAACCCTAAAGGGAGCCCTTTTTTTGAACAATAT TACCG 133GGAATTAGAGCTTTTTTTTCAGACCAGGCGCGTTGGGAAGATTTTTT TTCCAGGCAAAGC 134AATCATGGTCATTTTTTTTTTTGCCCGAACTCAGGTTTAACTTTTTTT TCAGTATGTTAG 135TTTCATTGAGTAGATTTAGTTTCTATATTT 136 AACAGTTAGGTCTTTACCCTGATCCAACAG 137GTGAATATAGTAAATTGGGCTTTAATGCAG 138 CTCAGCAGGCTACAGAGGCTTTAACAAAGT 139GTTAGTAACTTTCAACAGTTTCAAAGGCTC 140 GTACCAGGTATAGCCCGGAATAGAACCGCC 141GCCAGCAGCCTTGATATTCACAAACGGGGT 142 TAGAAAAGGCGACATTCAACCGCAGAATCA 143ATCCCAAAAAAATGAAAATAGCAAGAAACA 144 CTTATCACTCATCGAGAACAAGCGGTATTC 145CCAGTATGAATCGCCATATTTAGTAATAAG 146 GCTTAGAATCAAAATCATAGGTTTTAGTTA 147ATTATCAGTTTGGATTATACTTGCGCAGAG 148 ATGCGCGTACCGAACGAACCACGCAAATCA 149TTAACCGTCACTTGCCTGAGTACTCATGGA 150 GGAAGGGGGCAAGTGTAGCGGTGCTACAGG 151CTGGTTTGTTCCGAAATCGGCATCTATCAG 152 GTGCTGCCCCAGTCACGACGTTTGAGTGAG 153CAGGAAGTAATATTTTGTTAAAAACGGCGG 154 CCTTTATCATATATTTTAAATGGATATTCA 155CCCCAGCGGGAACGAGGCGCAGACTATTCATT Analyte-Binding Oligonucleotides 156AACCGAGGGCAAAGACACCACGGATAAATATTTTTCACTCACCTCCATCTCCACTCCTACCCATCCAACTCCCAC Surface-Binding Oligonucleotides 157GTCAGGAAGAGGTCATTTTTGCTCTGGAAGTTTTACCATCTTCCTC TCCAC 158ATACATACAACACTATCATAACATGCTTTATTTTACCATCTTCCTC TCCAC 159ACAACGGAAATCCGCGACCTGCCTCATTCATTTTACCATCTTCCTC TCCAC 160CAAAAGGTTCGAGGTGAATTTCTCGTCACCTTTTACCATCTTCCTC TCCAC 161ATTTCATGACCGTGTGATAAATAATTCTTATTTTACCATCTTCCTC TCCAC 162GCGAATTATGAAACAAACATCATAGCGATATTTTACCATCTTCCT CTCCAC 163ACAGTTGTTAGGAGCACTAACATATTCCTGTTTTACCATCTTCCTC TCCAC 164AATACCTATTTACATTGGCAGAAGTCTTTATTTTACCATCTTCCTC TCCAC 165CCATGTACCGTAACACTGTAGCATTCCACAGATTCCAGACTTTTAC CATCTTCCTCTCCAC 166CTAAACAGGAGGCCGATAATCCTGAGAAGTGTCACGCAAATTTTA CCATCTTCCTCTCCAC 167CAGTGCCCCCCCTGCCTATTTCTTTGCTCATTTTACCATCTTCCTCT CCAC 168AGTTTGCGCATTTTCGGTCATAGAGCCGCCTTTTACCATCTTCCTC TCCAC 169ATGAAATGAAAAGTAAGCAGATACAATCAATTTTACCATCTTCCT CTCCAC 170TAAGAACGGAGGTTTTGAAGCCTATTATTTTTTTACCATCTTCCTC TCCAC 171GGCGATGTTTTTGGGGTCGAGGGCGAGAAATTTTACCATCTTCCT CTCCAC 172CTAACTCCCAGTCGGGAAACCTGGTCCACGTTTTACCATCTTCCTC TCCAC 173ATTGACCCGCATCGTAACCGTGAGGGGGATTTTTACCATCTTCCTC TCCAC 174ACCGTTCATTTTTGAGAGATCTCCCAAAAATTTTACCATCTTCCTC TCCAC 175AGCTAATGCAGAACGCGAGAAAAATAATATCCTGTCTTTCTTTTAC CATCTTCCTCTCCAC 176AATCATACAGGCAAGGCAGAGCATAAAGCTAAGGGAGAAGTTTTA CCATCTTCCTCTCCACLabel-Binding Oligonucleotides 177TTTGGTGGCATCAATTCTAGGGCGCGAGCTGAAAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 178TTTTCCCAATTCTGCGAACCCATATAACAGTTGATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 179TTTATTGCTCCTTTTGATATTAGAGAGTACCTTTATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 180TTTCCATAAATCAAAAATCCAGAAAACGAGAATGATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 181TTTCGAGGCATAGTAAGAGACGCCAAAAGGAATTATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 182TTTGAAACACCAGAACGAGAGGCTTGCCCTGACGATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 183TTTCTGATAAATTGTGTCGAGATTTGTATCATCGCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 184TTTGAACGAGGGTAGCAACGCGAAAGACAGCATCGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 185TTTGGTTTATCAGCTTGCTAGCCTTTAATTGTATCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 186TTTGGGATTTTGCTAAACAAATGAATTTTCTGTATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 187TTTACAAACTACAACGCCTGAGTTTCGTCACCAGTTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 188TTTAGCCACCACCCTCATTGAACCGCCACCCTCAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 189TTTGAGAGGGTTGATATAAGCGGATAAGTGCCGTCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 190TTTGTATAAACAGTTAATGTTGAGTAACAGTGCCCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 191TTTGCAGGTCAGACGATTGTTGACAGGAGGTTGAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 192TTTTAGCGCGTTTTCATCGCTTTAGCGTCAGACTGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 193TTTGCGCCAAAGACAAAAGTTCATATGGTTTACCATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 194TTTCCGAAGCCCTTTTTAAAGCAATAGCTATCTTATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 195TTTTTTTTTGTTTAACGTCTCCAAATAAGAAACGATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 196TTTAACCTCCCGACTTGCGGCGAGGCGTTTTAGCGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 197TTTTAAACCAAGTACCGCATTCCAAGAACGGGTATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 198TTTAGATAAGTCCTGAACACCTGTTTATCAACAATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 199TTTGTAAAGTAATTCTGTCAAAGTACCGACAAAAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 200TTTAGTAGGGCTTAATTGAAAAGCCAACGCTCAACTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 201TTTAATGGTTTGAAATACCCTTCTGACCTAAATTTTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 202TTTAGTCAATAGTGAATTTTTAAGACGCTGAGAAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 203TTTTGAGCAAAAGAAGATGATTCATTTCAATTACCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 204TTTCAATATAATCCTGATTGATGATGGCAATTCATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 205TTTGTTATCTAAAATATCTAAAGGAATTGAGGAAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 206TTTACATCGCCATTAAAAAAACTGATAGCCCTAAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 207TTTTCGTCTGAAATGGATTACATTTTGACGCTCAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 208TTTTTGATTAGTAATAACATTGTAGCAATACTTCTTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 209TTTAGGAACGGTACGCCAGTAAAGGGATTTTAGACTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 210TTTGAGCACGTATAACGTGCTATGGTTGCTTTGACTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 211TTTCGGGCGCTAGGGCGCTAAGAAAGCGAAAGGAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 212TTTATCACCCAAATCAAGTGCCCACTACGTGAACCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 213TTTATCCTGTTTGATGGTGGCCCCAGCAGGCGAAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 214TTTGCTCACTGCCCGCTTTACATTAATTGCGTTGCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 215TTTGTAACGCCAGGGTTTTAAGGCGATTAAGTTGGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 216TTTCGTTGGTGTAGATGGGGTAATGGGATAGGTCATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 217TTTTTTAAATTGTAAACGTATTGTATAAGCAAATATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 218TTTGCCGGAGAGGGTAGCTTAGCTGATAAATTAATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 219TTTAAATTTTTAGAACCCTTTCAACGCAAGGATAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 220TTTTAAGCAATAAAGCCTCAAAGAATTAGCAAAATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC

Example 9 SNAP Synthesis and Purification

SNAPs were synthesized via the method described in Example 7.Synthesized SNAPs were designed to be substantially square DNA origamistructures with an approximately 83 nanometer (nm) edge length. Eachsquare SNAP contained 109 oligonucleotides with pendant handles forbinding of additional components to a SNAP via complementaryoligonucleotide conjugation to pendant groups: 1 pendant single-strandedDNA handle for coupling an analyte to an upper display face, 64 pendantsingle-stranded DNA handles for coupling a SNAP to a surface, and 44discrete, pendant single-stranded DNA handles for coupling detectablefluorescent labels to the 4 edges of the SNAP (11 per side). Alloligonucleotide sequences were designed using CADNANO2 software.

Table I contains sequence listings for coupling regions of SNAPoligonucleotides. SEQ. ID 1 is the sequence listing for the couplingregion of an oligonucleotide that is configured to couple to acomplementary oligonucleotide that is conjugated to an analyte. SEQ. ID2 is the sequence listing for the coupling region of an oligonucleotidethat is configured to couple to a complementary oligonucleotide that isconjugated to the surface of a solid support. SEQ. ID 3 is the sequencelisting for the coupling region of an oligonucleotide that is configuredto couple to a complementary oligonucleotide that is conjugated to afluorescent Alexa-Fluor™ 488 dye molecule. The sequences listed in TableII were each designed to exclude the nucleotide guanosine, therebyavoiding the likelihood of self-complementarity (i.e., the formation ofsecondary structures). It was expected that pendant single-stranded DNAsurface-interacting moieties (e.g., SEQ ID 2) would be more likely tobind to complementary, surface-linked oligonucleotides at ambienttemperatures (e.g., about 20° C.) if no secondary structures werepresent

Table III contains sequence listings for the 217 staple oligonucleotidesutilized to form the SNAPs with 64 pendant surface-linked moieties.Pendant regions of the 65 coupling oligonucleotides are highlighted inbold text. All staple oligonucleotides listed in Table IV were combinedwith M13mp18 single-stranded phage genomic DNA to fold the DNA origamistructure.

TABLE IV SEQ. ID 5′-3′ DNA Sequence Listing 221 TCATTTGCTAATAGTAGTAGCATT222 TTTCATTGAGTAGATTTAGTTTCTATATTT 223 AACAGTTAGGTCTTTACCCTGATCCAACAG224 TAGAGCTTCAGACCGGAAGCAAACCTATTATA 225AGATTTAGACGATAAAAACCAAAAATCGTCAT 226 CATTATTAGCAAAAGAAGTTTTGC 227GTGAATATAGTAAATTGGGCTTTAATGCAG 228 AGGCTTTTCAGGTAGAAAGATTCAATTACC 229ACCAGACGGAATACCACATTCAACGAGATGGT 230 AAAAGAATAACCGAACTGACCAACTTCATCAA231 CCCCAGCGGGAACGAGGCGCAGACTATTCATT 232 CTCAGCAGGCTACAGAGGCTTTAACAAAGT233 CATAAGGGACACTAAAACACTCACATTAAA 234 ACTTAGCCATTATACCAAGCGCGAGAGGACTA235 TTTCACGTCGATAGTTGCGCCGACCTTGCAGG 236 CGGGTAAAATTCGGTCGCTGAGGAATGACA237 GTTAGTAACTTTCAACAGTTTCAAAGGCTC 238 CTTGATACTGAAAATCTCCAAAAAAGCGGAGT239 ACCCTCATTCAGGGATAGCAAGCC 240 GTACCAGGTATAGCCCGGAATAGAACCGCC 241GCCAGCAGCCTTGATATTCACAAACGGGGT 242 TTATTCTGACTGGTAATAAGTTTTAACAAATA 243GACGGAAAACCATCGATAGCAGCATTGCCATCTTTTCATACACCCTCA 244AGGCCGGAACCAGAGCCACCACCG 245 TAGAAAAGGCGACATTCAACCGCAGAATCA 246TCACCGGAAACGTCACCAATGAATTATTCA 247 ATTAGCGTCCGTAATCAGTAGCGAATTGAGGG 248CGCTAATAGGAATACCCAAAAGAAATACATAA 249TGCCAGTTATAACATAAAAACAGGACAAGAATTGAGTTAACAGAAGG A 250ATCCCAAAAAAATGAAAATAGCAAGAAACA 251 ATAATAACTCAGAGAGATAACCCGAAGCGC 252AAAGTTACGCCCAATAATAAGAGCAGCCTTTA 253 ATTAGACGGAGCGTCTTTCCAGAGCTACAA 254CTTATCACTCATCGAGAACAAGCGGTATTC 255 AGATTAGTATATAGAAGGCTTATCCAAGCCGT 256AGAATATCAGACGACGACAATAAA 257 CCAGTATGAATCGCCATATTTAGTAATAAG 258GCTTAGAATCAAAATCATAGGTTTTAGTTA 259 CTTTGAATTACATTTAACAATTTCTAATTAAT 260CGGGAGAATTTAATGGAAACAGTA 261 CTACCTTTAGAATCCTTGAAAACAAGAAAACA 262ATTATCAGTTTGGATTATACTTGCGCAGAG 263 TTACCTTTACAATAACGGATTCGCAAAATT 264GCATCACCAGTATTAGACTTTACAGTTTGAGT 265 CCTCAATCCGTCAATAGATAATACAGAAACCA266 TGGAAGGGAGCGGAATTATCATCAACTAATAG 267AGACAATAAGAGGTGAGGCGGTCATATCAAAC 268 ATGCGCGTACCGAACGAACCACGCAAATCA 269GATTTAGATTGCTGAACCTCAAAGTATTAA 270 CACCGCCTGAAAGCGTAAGAATACATTCTG 271GATAAAACTTTTTGAATGGCTATTTTCACCAG 272 TTAACCGTCACTTGCCTGAGTACTCATGGA 273GCGCGTACTTTCCTCGTTAGAATC 274 GGAAGGGGGCAAGTGTAGCGGTGCTACAGG 275CTGGTTTGTTCCGAAATCGGCATCTATCAG 276 AGCACTAAAAAGGGCGAAAAACCGAAATCCCT 277AAGTGTAATAATGAATCGGCCAACCACCGCCT 278 AATTCCACGTTTGCGTATTGGGCG 279GTGCTGCCCCAGTCACGACGTTTGAGTGAG 280 GAGAGGCGACAACATACGAGCCGCTGCAGG 281AGCTGCATAGCCTGGGGTGCCTAAGTAAAACG 282 TTCATCAACGCACTCCAGCCAGCTGCTGCGCA283 CCCGTCGGGGGACGACGACAGTATCGGGCCTC 284 CAGGAAGTAATATTTTGTTAAAAACGGCGG285 AGGAAGATCATTAAATGTGAGCGTTTTTAA 286 AGTTTGAGATTCTCCGTGGGAACAATTCGCAT287 AGACAGTCCATTGCCTGAGAGTCTTCATATGT 288 CCAATAGGAAACTAGCATGTCAAGGAGCAA289 CCTTTATCATATATTTTAAATGGATATTCA 290 TATCAGGTAAATCACCATCAATATCAATGCCT291 ACTTTTGCATCGGTTGTACTTTTTTTAACCTGTTTAGGACCATTA 292GAGTAATGTGTAGGTAAAGATTTTTTGTTTTAAATATG 293ACAAGAGTTTTTTTCGCGTTTTAATTCAAAAAGA 294 CAAAAATAATTTTTTTTGTTTAGAC 295CCGCTTCTGGTTTTTTCGTTAATAAAACGAACTAAATTATACC 296GAACCGCCTCTTTACCTAAAACGAAAGAGGC 297 AGAACCGCATTTACCGTTTTACCGATATATACGTAA298 AGGAGTGTAAACATGAAAGTATTAAGAGGCTTTTTTTGCGAATAATAA TTT 299ACCGTACTCAGGTTTTTGATCTAAAGTTT 300TGTCGTCTCAGCCCTCATATTTTTTTCGCCACCCTCAGGTGTATC 301GAGAATAGAAAGGAACAACTATTTTCTCAAGAGAAGGA 302ACAACCATTTTTTCATACATGGCTTTTAAGCGCA 303 TGCCACTACTTTTTTTGCCACCCTC 304AGGACAGATGATTTTTTCACCAGTAGCACCATTACCGACTTGA 305AATCATGGTCATTTTTTTTTTTGCCCGAACTCAGGTTTAACTTTTTTTTC AGTATGTTAG 306CATAAATCAATTTAGTCAGAGGGTAATTGAG 307 TTGCTTCTTATATGTATTTTACGCTAACGGAGAATT308 GCGAGAAAATAAACACCGGAATCATAATTATTTTTTTCGCCCAATAGC AAG 309AACATGTAATTTTTTTTGAAACCAATCAA 310TAATCGGCCATCCTAATTTTTTTTTTTTTTCGAGCCAACAACGCC 311TTTTATTTTCATCGTAGGAATTTTTAGCCTGTTTAGTA 312TTTTATCTTTTTTATCCAATCGCAAGAGTTGGGT 313 AACTGAACATTTTTTTTGAATAACC 314ATTAAGACTCCTTTTTAATATACAGTAACAGTACCGAAATTGC 315CCAGGGTGGTTTTGCAAATGAAAAATCTAAA 316 ACGGGCAAGTTCCAGTTTTTTCTGACCTGCAACAGT317 CCAACGTCATCGGAACCCTAAAGGGAGCCCTTTTTTTGAACAATATTA CCG 318GCGTAACCACCATTTTTGAGTAAAAGAGT 319CTGTCCATTTTTATAATCATTTTTTTCTTAATGCGCCCACGCTGC 320CAAACTATCGGCCTTGCTGGTTTTTGAGCTTGACGGGG 321GCCAACATTTTTTCCACTATTAAAGAAATAGGGT 322 GCCACGCTGTTTTTTTACCAGTGAG 323GACAACTCGTATTTTTTCCTGTGTGAAATTGTTATCCGAGCTC 324GGAATTAGAGCTTTTTTTTCAGACCAGGCGCGTTGGGAAGATTTTTTTT CCAGGCAAAGC 325CAGAGGGGGTTTTGCCTTCCTGTAGCCAGCT 326 TGGATAGCAAGCCCGATTTTTAATCGTAAACGCCAT327 AAGCGAACAATTGCTGAATATAATGCTGTATTTTTTTGTGAGAAAGGC CGG 328GATACATTTCGCTTTTTTGACCCTGTAAT Analyte-Binding Oligonucleotides 329AACCGAGGGCAAAGACACCACGGATAAATATTTTTCACTCACCTCCATCTCCACTCCTACCCATCCAACTCCCAC Surface-Binding Oligonucleotides 330GTCAGGAAGAGGTCATTTTTGCTCTGGAAGTTTTACCATCTTCCTCTC CAC 331CAACTAAAGTACGGTGGGATGGCTTTTTACCATCTTCCTCTCCAC 332AAATATTCCAAAGCGGATTGCATCGAGCTTCATTTTACCATCTTCCTC TCCAC 333ATACATACAACACTATCATAACATGCTTTATTTTACCATCTTCCTCTC CAC 334TTAAGAGGGTCCAATACTGCGGATAGCGAGTTTTACCATCTTCCTCTCC AC 335GTCAGAAGATTGAATCCCCCTCAACCTCGTTTTTTTACCATCTTCCTC TCCAC 336GAGTAATCTTTTAAGAACTGGCTCCGGAACAATTTTACCATCTTCCTC TCCAC 337ACCCAAATAACTTTAATCATTGTGATCAGTTGTTTTACCATCTTCCTC TCCAC 338ACAACGGAAATCCGCGACCTGCCTCATTCATTTTACCATCTTCCTCTC CAC 339AGTCAGGACATAGGCTGGCTGACCTTTGAAAGTTTTACCATCTTCCTC TCCAC 340TTATGCGATTGACAAGAACCGGAGGTCAATTTTTACCATCTTCCTCTC CAC 341TTAATTTCCAACGTAACAAAGCTGTCCATGTTTTTTACCATCTTCCTC TCCAC 342GAGTTAAATTCATGAGGAAGTTTCTCTTTGACTTTTACCATCTTCCTC TCCAC 343CAAAAGGTTCGAGGTGAATTTCTCGTCACCTTTTACCATCTTCCTCTC CAC 344AAGACTTTGGCCGCTTTTGCGGGATTAAACAGTTTTACCATCTTCCTC TCCAC 345CCATGTACCGTAACACTGTAGCATTCCACAGATTCCAGACTTTTACCA TCTTCCTCTCCAC 346CAGTGCCCCCCCTGCCTATTTCTTTGCTCATTTTACCATCTTCCTCTCC AC 347TTAGGATTAGCGGGGTGGAACCTATTTTACCATCTTCCTCTCCAC 348GAGCCGCCTTAAAGCCAGAATGGAGATGATACTTTTACCATCTTCCT CTCCAC 349AGTTTGCGCATTTTCGGTCATAGAGCCGCCTTTTACCATCTTCCTCTC CAC 350GTCTCTGACACCCTCAGAGCCACATCAAAATTTTACCATCTTCCTCTC CAC 351AATCCTCAACCAGAACCACCACCAGCCCCCTTTTTTACCATCTTCCTC TCCAC 352AGGTGGCAGAATTATCACCGTCACCATTAGCATTTTACCATCTTCCTC TCCAC 353ATGAAATGAAAAGTAAGCAGATACAATCAATTTTACCATCTTCCTCT CCAC 354GCCATTTGCAAACGTAGAAAATACCTGGCATGTTTTACCATCTTCCTC TCCAC 355TTAAAGGTACATATAAAAGAAACAAACGCATTTTACCATCTTCCTCT CCAC 356AGGGAAGGATAAGTTTATTTTGTCAGCCGAACTTTTACCATCTTCCTC TCCAC 357CAAATCAGTGCTATTTTGCACCCAGCCTAATTTTTTACCATCTTCCTC TCCAC 358TAAGAACGGAGGTTTTGAAGCCTATTATTTTTTTACCATCTTCCTCTC CAC 359CAGAGAGAACAAAATAAACAGCCATTAAATCATTTTACCATCTTCCT CTCCAC 360AGCTAATGCAGAACGCGAGAAAAATAATATCCTGTCTTTCTTTTACCA TCTTCCTCTCCAC 361ATTTCATGACCGTGTGATAAATAATTCTTATTTTACCATCTTCCTCTC CAC 362TCATATGCGTTATACAAAGGCGTTTTTTACCATCTTCCTCTCCAC 363TTTCCCTTTTAACCTCCGGCTTAGCAAAGAACTTTTACCATCTTCCTC TCCAC 364AAATAAGAACTTTTTCAAATATATCTGAGAGATTTTACCATCTTCCTC TCCAC 365GCGAATTATGAAACAAACATCATAGCGATATTTTACCATCTTCCTCT CCAC 366TATATAACGTAAATCGTCGCTATATTTGAATTTTACCATCTTCCTCTC CAC 367AACATTATGTAAAACAGAAATAAATTTTACATTTTTACCATCTTCCTC TCCAC 368CCAGAAGGTTAGAACCTACCATATCCTGATTGTTTTACCATCTTCCTC TCCAC 369AAATTAATACCAAGTTACAAAATCCTGAATAATTTTACCATCTTCCTC TCCAC 370ACAGTTGTTAGGAGCACTAACATATTCCTGTTTTACCATCTTCCTCTC CAC 371GTAGATTTGTTATTAATTTTAAAAAACAATTCTTTTACCATCTTCCTC TCCAC 372ATTTGCACCATTTTGCGGAACAAATTTGAGTTTTACCATCTTCCTCTC CAC 373ATTAGAGCAATATCTGGTCAGTTGCAGCAGAATTTTACCATCTTCCTC TCCAC 374CCAGCCATCCAGTAATAAAAGGGACGTGGCACTTTTACCATCTTCCT CTCCAC 375AATACCTATTTACATTGGCAGAAGTCTTTATTTTACCATCTTCCTCTC CAC 376TCACACGATGCAACAGGAAAAACGGAAGAACTTTTTACCATCTTCCT CTCCAC 377CTAAACAGGAGGCCGATAATCCTGAGAAGTGTCACGCAAATTTTACC ATCTTCCTCTCCAC 378GGCGATGTTTTTGGGGTCGAGGGCGAGAAATTTTACCATCTTCCTCT CCAC 379AAAGCCGGCGAACGTGTGCCGTAATTTTACCATCTTCCTCTCCAC 380GGCCCTGAAAAAGAATAGCCCGAGCGTGGACTTTTTACCATCTTCCT CTCCAC 381CTAACTCCCAGTCGGGAAACCTGGTCCACGTTTTACCATCTTCCTCTC CAC 382TGAGTGTTCAGCTGATTGCCCTTGCGCGGGTTTTACCATCTTCCTCTC CAC 383TATAAATCGAGAGTTGCAGCAAGCGTCGTGCCTTTTACCATCTTCCTC TCCAC 384ACTGTTGGAGAGGATCCCCGGGTACCGCTCACTTTTACCATCTTCCTC TCCAC 385TTCGCTATTGCCAAGCTTGCATGCGAAGCATATTTTACCATCTTCCTC TCCAC 386ATTGACCCGCATCGTAACCGTGAGGGGGATTTTTACCATCTTCCTCTC CAC 387GAATTCGTGCCATTCGCCATTCAGTTCCGGCATTTTACCATCTTCCTC TCCAC 388TCGACTCTGAAGGGCGATCGGTGCGGCCTCTTTTACCATCTTCCTCTC CAC 389ACGGCCAGTACGCCAGCTGGCGAACATCTGCCTTTTACCATCTTCCTC TCCAC 390ACCCCGGTTGTTAAATCAGCTCATAGTAACAATTTTACCATCTTCCTC TCCAC 391ACCGTTCATTTTTGAGAGATCTCCCAAAAATTTTACCATCTTCCTCTC CAC 392TAAATTTTTGATAATCAGAAAAGCACAAAGGCTTTTACCATCTTCCTC TCCAC 393AATCATACAGGCAAGGCAGAGCATAAAGCTAAGGGAGAAGTTTTACC ATCTTCCTCTCCACLabel-Binding Oligonucleotides 394TTTGGTGGCATCAATTCTAGGGCGCGAGCTGAAAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 395TTTTCCCAATTCTGCGAACCCATATAACAGTTGATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 396TTTATTGCTCCTTTTGATATTAGAGAGTACCTTTATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 397TTTCCATAAATCAAAAATCCAGAAAACGAGAATGATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 398TTTCGAGGCATAGTAAGAGACGCCAAAAGGAATTATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 399TTTGAAACACCAGAACGAGAGGCTTGCCCTGACGATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 400TTTCTGATAAATTGTGTCGAGATTTGTATCATCGCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 401TTTGAACGAGGGTAGCAACGCGAAAGACAGCATCGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 402TTTGGTTTATCAGCTTGCTAGCCTTTAATTGTATCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 403TTTGGGATTTTGCTAAACAAATGAATTTTCTGTATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 404TTTACAAACTACAACGCCTGAGTTTCGTCACCAGTTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 405TTTAGCCACCACCCTCATTGAACCGCCACCCTCAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 406TTTGAGAGGGTTGATATAAGCGGATAAGTGCCGTCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 407TTTGTATAAACAGTTAATGTTGAGTAACAGTGCCCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 408TTTGCAGGTCAGACGATTGTTGACAGGAGGTTGAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 409TTTTAGCGCGTTTTCATCGCTTTAGCGTCAGACTGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 410TTTGCGCCAAAGACAAAAGTTCATATGGTTTACCATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 411TTTCCGAAGCCCTTTTTAAAGCAATAGCTATCTTATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 412TTTTTTTTTGTTTAACGTCTCCAAATAAGAAACGATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 413TTTAACCTCCCGACTTGCGGCGAGGCGTTTTAGCGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 414TTTTAAACCAAGTACCGCATTCCAAGAACGGGTATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 415TTTAGATAAGTCCTGAACACCTGTTTATCAACAATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 416TTTGTAAAGTAATTCTGTCAAAGTACCGACAAAAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 417TTTAGTAGGGCTTAATTGAAAAGCCAACGCTCAACTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 418TTTAATGGTTTGAAATACCCTTCTGACCTAAATTTTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 419TTTAGTCAATAGTGAATTTTTAAGACGCTGAGAAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 420TTTTGAGCAAAAGAAGATGATTCATTTCAATTACCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 421TTTCAATATAATCCTGATTGATGATGGCAATTCATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 422TTTGTTATCTAAAATATCTAAAGGAATTGAGGAAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 423TTTACATCGCCATTAAAAAAACTGATAGCCCTAAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 424TTTTCGTCTGAAATGGATTACATTTTGACGCTCAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 425TTTTTGATTAGTAATAACATTGTAGCAATACTTCTTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 426TTTAGGAACGGTACGCCAGTAAAGGGATTTTAGACTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 427TTTGAGCACGTATAACGTGCTATGGTTGCTTTGACTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 428TTTCGGGCGCTAGGGCGCTAAGAAAGCGAAAGGAGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 429TTTATCACCCAAATCAAGTGCCCACTACGTGAACCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 430TTTATCCTGTTTGATGGTGGCCCCAGCAGGCGAAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 431TTTGCTCACTGCCCGCTTTACATTAATTGCGTTGCTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 432TTTGTAACGCCAGGGTTTTAAGGCGATTAAGTTGGTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 433TTTCGTTGGTGTAGATGGGGTAATGGGATAGGTCATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 434TTTTTTAAATTGTAAACGTATTGTATAAGCAAATATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 435TTTGCCGGAGAGGGTAGCTTAGCTGATAAATTAATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 436TTTAAATTTTTAGAACCCTTTCAACGCAAGGATAATTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC 437TTTTAAGCAATAAAGCCTCAAAGAATTAGCAAAATTTTAACTACTCCCACTCTCACCCTCACCCTACTCCAACTCAAC

Example 10 Deposition of SNAPs on Prepared Surfaces

Surfaces were prepared for the formation of unpatterned arrays. A layerof (3-aminopropyl) trimethoxysilane (APTMS) was deposited on the surfaceof a glass slide. The APTMS-coated surface was subsequently reacted withazide-PEG-NHS ester to covalently form a PEG passivating layer on thesurface of the glass slide. After forming the PEG passivating layer,surface-linked azide groups were conjugated to oligonucleotidescontaining a dibenzylcyclooctyne (DBCO) functional groups. Eacholigonucleotide had the sequence 5′-DBCO-TGTGGAGAGGAAGATGGTA-3′ (SEQ. ID438). A reactive scheme for the preparation of the glass surface isshown in FIG. 42 . Oligonucleotide arrays were formed with varyingsurface oligonucleotide densities by varying the concentration ofoligonucleotide contacted with the azide-containing surface.Oligonucleotide concentrations of 0.01 micromolar (μM), 0.1 μM, and 1 μMwere utilized for surface preparation.

Prepared glass surfaces were contacted with DNA origami containing 20surface-interacting moieties, as described in Example 8. 44 Alexa-Fluor488 fluorescent dyes were bound to each DNA origami via a complementaryoligonucleotide to the pendant region of the label-bindingoligonucleotides (see SEQ. ID 3). Two polypeptides were bound to eachDNA origami via complementary oligonucleotides to the pendant regions ofanalyte-binding oligonucleotides (see SEQ. ID 1, for example). Eachpolypeptide was a 12-amino acid histidine peptide (SEQ. ID439—HHHHHHHHHHHH), hereafter referred to as His-12.

DNA origami containing the pendant oligonucleotides were deposited onthe prepared glass surface by hybridization of pendantsurface-interacting oligonucleotides (see SEQ. ID 2) to thesurface-linked oligonucleotides (see SEQ. ID 438). The deposition bufferis described in Example 3. Deposition of His-12 DNA origami wasperformed for four separate arrays. Two additional arrays were preparedwith DNA origami containing the pendant oligonucleotides but nopolypeptides (control SNAPs). Arrays were formed using oligonucleotides

After array formation, SNAP locations on each array were identified byfluorescence microscopy imaging at 488 nm. After determining theposition of deposited SNAPs on each array, arrays were contacted withhistidine-binding detectable probes. Each detectable probe comprised aDNA origami tile with 20 coupled B1 aptamers and 44 conjugatedAlexa-Fluor 647 fluorescent dyes. Probes were contacted with each arrayat a concentration of 30 nM and were incubated for 30 minutes. Unboundprobes were rinsed from each array by a rinse buffer (see Example 3).After rinsing, each array was imaged to identify array addresses wherethe B1 probes were bound.

FIG. 43 shows binding data for the B1 probes against each array ofSNAPs. Binding of the B1 probe was observed for at least 20-25% of arrayaddresses. In contrast, near-zero binding of B1 probe topolypeptide-free SNAPs was observed. FIG. 46 shows fluorescentmicroscopy image data for SNAPs deposited on oligonucleotide-containingsurfaces containing differing surface densities of oligonucleotides.SNAPs were contacted with the oligonucleotide-containing surfaces at aconcentration of 10 picomolar (pM) or 100 pM. Deposited SNAP densitieswere observed to increase with increased oligonucleotide surface densityand increased SNAP concentration.

Example 11 Detection of Polypeptides on SNAPs

Two arrays of SNAPs were prepared via the method described in Example10. Each array was prepared with SNAPs that contained 20 pendant captureoligonucleotides and a single polypeptide coupling oligonucleotide. EachSNAP was coupled to a single His-12 peptide. After array preparation,each array was incubated with B1 probes as described in Example 10. Theprobes were contacted with each array at a concentration of 10 nM for 20minutes. Probe binding was detected via fluorescence microscopy at 647nm. FIG. 44 depicts the fraction of observed array addresses withdetected B1 probe binding. About 10% of array addresses were observed tobind B1 probes.

Example 12 Detection of Polypeptides on SNAPs

Oligonucleotide-containing glass surfaces were prepared according to thescheme of FIG. 42 . Additional glass surfaces were prepared containingonly APTMS surface-linked moieties. SNAPs were prepared with 20 pendant,capture moieties, as described in Example 9. Each SNAP was configured tohave two polypeptide binding sites. SNAPs were conjugated tostreptavidin polypeptides, with each streptavidin having 2 His-12 tags.

SNAPs were incubated with prepared glass surfaces to form polypeptidearrays. A total of 6 replicates of each type of surface(APTMS-containing and oligo-containing) were tested, with 4 surfacesbeing incubated with streptavidin-conjugated SNAPs and 2 surfaces beingincubated with SNAPs containing no polypeptides.

After SNAP deposition, each glass surface was imaged by confocalfluorescent microscopy to identify arrays addresses for deposited SNAPs.Imaging of SNAP addresses was performed by detection of Alexa-Fluor 488dyes on each SNAP. After identifying occupied array addresses, SNAPswere contacted with B1 aptamer probes, as described in Examples 10 and11. Probe binding was detected by confocal fluorescent microscopy bydetection of Alexa-Fluor 647 dyes on each probe. 647 nm data wascompared to 488 nm data to determine a fraction of occupied arrayaddresses that were observed to bind a B1 probe. FIG. 45 displaysbinding detection data for SNAPs deposited on APTMS surfaces andoligonucleotide-containing surfaces. APTMS surfaces were observed tohave a lower binding detection rate of His-12 containing-polypeptides,and a higher false positive rate (detection of SNAPs containing nopolypeptides). Oligonucleotide-containing surfaces were observed to havea higher binding detection rate of His-12 containing polypeptides, and alower false positive rate. The presence of the PEG passivating layer andthe increased specificity of surface interactions between the SNAPs andthe oligonucleotide-containing surfaces may have increased thelikelihood of true-positive detection and decreased the likelihood offalse positive detection.

Example 13 Formation of Unpatterned SNAP Arrays

SNAPs were deposited on unpatterned glass surfaces containing PEG-azidesurface-linked moieties. The glass surfaces were prepared according tothe scheme shown in FIG. 42 , with the final oligonucleotide conjugationstep excluded. The surface concentration of azide groups was varied bymixing NHS-PEG2K-azide molecules and NHS-PEGSK molecules in differingratios. The ratio of NHS-PEG2K-azide to NHS-PEGSK molecules were variedbetween 5:95 and 100:0. After forming the azide-containing glasssurfaces, SNAPs containing a surface-coupling dibenzocyclooctylene(DBCO) moiety were contacted with the surfaces at a concentration of 1nanomolar (nM). SNAPs were incubated for at least 12 hours to facilitateformation of Click-type interactions between surface-linked azides andSNAP-coupled DBCO moieties. Incubations were performed at 20° C. and 4°C. to test the affect of temperature on deposition. Negative controlarrays were also formed by contacting azide-containing surfaces withSNAPs that did not have a DBCO moiety. FIG. 47 shows fluorescencemicroscopy images of SNAP arrays as a function of PEG2K-azide:PEG5Kratio and deposition temperature. Deposited SNAP concentrations on theunpatterned arrays were seen to increase with increasing surfacedensities of azide moieties and increasing temperature. In the absenceof a DBCO moiety coupled to a SNAP, minimal deposition of SNAPs wasobserved on glass surfaces.

Example 14 Synthesis and Characterization of SNAPs with PerviousStructures

Square, tile-shaped DNA origami comprising single-stranded DNA (ssDNA)were prepared by the method described in Example 7. The DNA origamistructure was folded from a mix of ssDNA oligos and the m13mp18 scaffoldssDNA. All the oligos including an oligo with a TCO display moiety weremixed in excess with the scaffold DNA. Purified DNA origami tiles weredeposited onto mica for AFM imaging (FIG. 59A). The measured tiledimensions matched the expected tile edge length (80-90 nm) and tileheight (2 nm).

After synthesis of the tile origami, a pervious structure was formed oneach DNA origami by TdT extension in the presence of an excess ofdeoxythymidine nucleotides, which extended ssDNA overhangs surroundingthe DNA origami tile seed structure. The pervious, poly-T ssDNAextensions are expected to lay substantially flat on a positivelycharged surface of a solid support. DNA origami tiles with poly-Textensions were imaged on mica with poly-lysine coating or amine (APTMS)covered glass surface (FIG. 59B). The DNA origami tile with poly-Textensions were found to have typical diameters in the range of 600-700nm; large enough to exclude the deposition of a second brushy tile on a400 nm size array site on a solid support.

According to AFM data, 95% of the DNA origami tile particles with poly-Textensions were intact (FIG. 59C). DNA origami tiles with poly-Textensions were coupled to an mTz-modified proteins according to themethod of Example 1. Analytical HPLC results showed that the fraction ofpoly-T extended DNA origami tiles with functional TCO groups was 95% andthe fraction with conjugated protein was 90% (FIG. 59C). FIG. 59D plotssize data for DNA origami in various configurations, includingorigami-only, with poly-T extensions in solution, and with poly-Textensions on a surface. The mean edge length of the compacted DNAorigami tiles was 90 nm. Dynamic light scattering measurements showedthat the mean diameter of the poly-T extended DNA origami in solutionwas 500 nm. Based on the AFM measurements, the mean diameter of poly-Textended DNA origami was 650 nm on the surface. In summary, poly-Textended DNA origami were conjugated to protein with high efficiency,and their large size is configured to prevent deposition of more thanone poly-T extended DNA origami at each site on a solid support.

Example 15 Single-Molecule Array Preparation

A patterned solid support was formed by photolithographic patterning ofa glass substrate. After photolithographic patterning, the solid supportwas functionalized with APTMS to provide a positively-charge surfacecoating. After APTMS deposition, the photolithographic photoresist wasstripped from the chip to provide a patterned array of binding sites (asshown in FIG. 67A). The patterning of the glass surface matched theexpected feature periodicity and spacing, and confirmed that only thepatterned features have the positively charged amine coating (FIG. 67B).The uniform intensity of the patterned regions demonstrated that theAPTMS coating was consistent within and between the features. Highresolution AFM characterization showed that the glass/silicon surfaceroughness was in the expected and workable range (<2 nm²) (FIG. 67C).The measured feature diameter (FIG. 67D) and pitch (FIG. 67E) matchedthe expected values of approximately 400 nanometers and 1.4 microns,respectively.

Example 16 Non-Poisson Array Loading with SNAPs Containing PerviousStructures

To assess single molecule occupancy on the chips, two versions of DNAorigami tiles with poly-T extensions are produced for mixingexperiments. DNA origami tiles with poly-T extensions are produced bythe method of Example 14, with a first version being labeled withAlexa-Fluor 488 dyes and a second version being labeled with Alexa-Fluor647 dyes. An equimolar mixture of the two types of SNAPs is deposited ona patterned glass array, as described in Example 15. By counting thefeatures lit up by a single wavelength (indicating only one depositedtile) and those lit up by both wavelengths (indicating more than onedeposited tile), it is possible to estimate single molecule occupancy.Double double-occupancy is observed at 5% of array sites for a 96%occupied array (i.e. 4% of sites containing no observed SNAPs). With noexclusion (Poisson deposition), it would be expected to observe nearly25% of the spots with double color. Atomic force microscopy (AFM) isalso used to demonstrate single molecule occupancy of array sites athigh resolution. AFM results suggest that 90% of the spots have a singlebrushy DNA origami tile.

To estimate a dynamic range afforded by an above-described arrayutilizing partially-structured SNAPs, dilution experiments and 488/647mixing experiments are used. At different dilution and 488-to-647 brushyDNA origami tile ratios, a number of observed 488 brushy tiles among 10⁵spots are determined. By extrapolating the data points, it isdemonstrated that a single DNA origami tile can be observed among 10⁷spots.

Example 17 Functional Nucleic Acids on SNAPs

An array of SNAPs was prepared to determine if a detectable label couldbe applied and removed from each SNAP on the array over multiple cyclesof binding and removal. A chip comprising a glass surface with a blanketlayer of (3-aminopropyl) trimethoxysilane (APTMS) was prepared. SNAPswere contacted with the APTMS-coated surface of the chip at aconcentration of 4.5 picomolar in a solution containing 1× Neoventuresbuffer, 0.1% Tween20, 0.001% lipidure, and 10 mM MgCl₂. Each SNAPcomprised a functional nucleic acid comprising a pendant single-strandedDNA coupled to a tile-shaped DNA origami. The functional nucleic acidhad a nucleotide sequence of ATTATACTACATACACC (SEQ. ID 440). TheSNAP-containing buffer was incubated on the APTMS-coated surface for 10minutes, then the surface was rinsed with a buffer comprising 1×Neoventures buffer, 0.1% Tween20, 0.001% lipidure, and 10 mM MgCl₂.

After preparing an array of randomly-deposited SNAPs on the APTMS-coatedsurface, the array underwent 14 detection cycles. Each detection cyclecomprised 1) contacting the array with a fluidic medium comprising afluorescently-labeled oligonucleotide with a nucleotide sequence ofTAATATGATGTATGTGG (SEQ. ID 441) and 5 Alexa-Fluor dyes, 2) incubatingthe fluorescently-labeled oligonucleotide with the array for 1 minute,3) rinsing the array with a solution containing 1× Neoventures buffer,0.1% Tween20, 0.001% lipidure, and 10 mM MgCl₂, 4) fluorescently imagingthe array to detect spatial locations of coupled fluorescently-labeledoligonucleotides, 5) applying a stripping buffer containing 6Mguanidinium hydrochloride and 10 mM MgCl₂, and 6) rinsing the array witha solution containing 1× Neoventures buffer, 0.1% Tween20, 0.001%lipidure, and 10 mM MgCl₂. Odd-numbered cycles (e.g., 1, 3, 5, . . . ,etc.) utilized a fluorescently-labeled oligonucleotide comprising anAlexa-Fluor 488 fluorophore, and even-numbered cycles (e.g., 2, 4, 6, .. . , etc.) utilized a fluorescently-labeled oligonucleotide comprisingan Alexa-Fluor 647 fluorophore.

FIG. 68 displays fluorescent imaging data for each cycle. Odd-numberedcycles are shown to have detection of fluorescence at array addresses inthe 488-nm channel of the fluorescent microscope, but virtually nodetection in the 647-nm channel of the fluorescent microscope.Even-numbered cycles are shown to have virtually no detection offluorescence at array addresses in the 488-nm channel of the fluorescentmicroscope, but have detection of fluorescence in the 647-nm channel ofthe fluorescent microscope. The results indicate that it is possibleto 1) strip an oligonucleotide detectable label from a functionalnucleic acid of a SNAP using a chaotropic agent (e.g., guanidiniumhydrochloride), and 2) not disrupt an electrostatic interaction betweena SNAP and a charge-surface when contacting the SNAP with a chaotropicagent.

An additional experiment was performed to assess the effect of a longernucleotide sequence on removal of the oligonucleotide under strippingconditions. Two arrays were prepared by the method described above. Thefirst array contained deposited SNAPs with a functional nucleic acidthat was configured to couple a fluorescently-labeled oligonucleotidewith a nucleotide sequence of ACAACTCAACCTCATCCCACTCCCACTCTCACCCTCATCAA(SEQ. ID 442). The second array comprised the SNAPs as described abovewith the functional nucleic acid with nucleotide sequenceTAATATGATGTATGTGG (SEQ. ID 441). The arrays were contacted with theirrespective fluorescently-labeled complementary oligonucleotides (eachcomplementary oligonucleotide containing 5 Alexa-Fluor 488 dyes),fluorescently imaged, incubated with 6M guanidinium chloride, thenre-contacted with their respective complementary oligonucleotides. FIG.70 displays fluorescent imaging data for the two arrays, depicting thefluorescent labeling of the functional nucleic acids, as well asstripping results for each respective array. The longer base-pairoligonucleotide is still detectable in many sites after the guanidiniumchloride incubation, suggesting that length of a functional nucleic acidsequence can be modulated to facilitate retention or removal of acomplementary oligonucleotide from the functional nucleic acid asnecessary.

Example 18 Multiplexed Arrays Utilizing Functional Nucleic Acids

An array of SNAPs was prepared via the method of Example 17. The mixtureof deposited SNAP comprises an equimolar mixture of a plurality of firsttile-shaped SNAPs with first functional nucleic acids, and a pluralityof second tile-shaped SNAPs with second functional nucleic acids. Thenucleotide sequence of the first functional nucleic acid wasATTATACTACATACACC (SEQ. ID 440), and the nucleotide sequence of thesecond functional nucleic acid was GTTTGTTGTTTGGGTTG (SEQ. ID 443).

The multiplexed array containing the first tile-shaped SNAPs and thesecond tile-shaped SNAPs was detected for 2 detection cycles, in whichthe first cycle utilized an Alexa-Fluor 488-labeled oligonucleotide witha sequence complementary to the first functional nucleic acid, and inwhich the second cycle utilized an Alexa-Fluor 488-labeledoligonucleotide with a sequence complementary to the second functionalnucleic acid. Each complementary oligonucleotide comprised 5 Alexa-Fluor488 dyes. FIGS. 69A and 69C display fluorescence microscopy images forthe binding of the first complementary oligonucleotide and the secondcomplementary oligonucleotide, respectively. FIGS. 69B and 69D displayfluorescence microscopy images for the post-application stripping inguanidinium hydrochloride of the first complementary oligonucleotide andthe second complementary oligonucleotide, respectively. As shown, theaddresses occupied by SNAPs of the first plurality of SNAPs can bedistinguished from addresses occupied by the second plurality of SNAPsbased upon the detection of binding of complementary oligonucleotides tofunctional nucleic acids of SNAPs.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.Notwithstanding the appended claims, the disclosure set forth herein isalso defined by the following clauses:

-   -   1. A composition, comprising:        -   a structured nucleic acid particle comprising (i) a display            moiety that is configured to couple to an analyte, (ii) a            capture moiety that is configured to couple with a surface;            and        -   (iii) a multifunctional moiety comprising a first functional            group and a second functional group;        -   wherein the multifunctional moiety is coupled to the            structured nucleic acid particle; and        -   wherein the first functional group is coupled to the display            moiety, and wherein the second functional group is coupled            to the capture moiety.    -   2. The composition of clause 1, wherein the multifunctional        moiety comprises a nucleic acid strand.    -   3. The composition of clause 1 or 2, wherein the structured        nucleic acid particle comprises a display face comprising the        display moiety and a capture face comprising the capture moiety.    -   4. The composition of clause 3, wherein the structured nucleic        acid particle comprises a plurality of tertiary structures,        wherein the display face comprises a first tertiary structure of        the plurality of tertiary structures, and the capture face        comprises a second tertiary structure of the plurality of        tertiary structures.    -   5. The composition of clause 4, wherein the first tertiary        structure is the same as the second tertiary structure.    -   6. The composition of clause 4, wherein the first tertiary        structure is different from the second tertiary structure.    -   7. The composition of any one of clauses 4-6, wherein the        nucleic acid strand is hybridized to the structured nucleic acid        particle, thereby forming a portion of the first tertiary        structure or a portion of the second tertiary structure.    -   8. The composition of clause 7, wherein the multifunctional        moiety is hybridized to the structured nucleic acid particle,        thereby forming a portion of the first tertiary structure and a        portion of the second tertiary structure.    -   9. The composition of any one of clause 4-8, wherein the        orientation of the display face or the orientation of the        capture face is defined relative to an axis of symmetry for the        first tertiary structure or an axis of symmetry for the second        tertiary structure.    -   10. The composition of clause 9, wherein the orientation of the        display face is the same as the orientation of the capture face.    -   11. The composition of clause 9, wherein the orientation of the        display face is offset from the orientation of the capture face        by at least about 90°.    -   12. The composition of clause 11, wherein the orientation of the        display face is offset from the orientation of the capture face        by about 180°.    -   13. The composition of any one of clauses 4-12, wherein the        display moiety comprises two or more display tertiary structures        of the plurality of tertiary structures.    -   14. The composition of any one of clauses 4-13, wherein the        capture moiety comprises two or more capture tertiary structures        of the plurality of tertiary structures.    -   15. The composition of clause 14, wherein a display tertiary        structure of the two or more display tertiary structures        comprises a capture tertiary structure of the two or more        capture tertiary structures.    -   16. The composition of clause 14, wherein the two or more        display tertiary structures do not comprise any capture tertiary        structure of the two or more capture tertiary structures.    -   17. The composition of clause 14, wherein the two or more        capture tertiary structures do not comprise any display tertiary        structure of the two or more display tertiary structures.    -   18. The composition of any one of clauses 2-17, wherein the        nucleic acid strand forms a hybridization region with the        structured nucleic acid particle, the hybridization region        comprising at least about 10 nucleotides.    -   19. The composition of clause 18, wherein the hybridization        region comprises at least about 20 nucleotides.    -   20. The composition of any one of clauses 2-19, wherein the        nucleic acid strand forms a hybridization region comprising at        least one helical revolution.    -   21. The composition of clause 20, wherein the hybridization        region comprises at least two helical revolutions.    -   22. The composition of any one of the preceding clauses, wherein        the structured nucleic acid particle comprises a scaffold strand        and a plurality of oligonucleotides hybridized to the scaffold        strand.    -   23. The composition of clause 22, wherein the scaffold strand        hybridized to the plurality of oligonucleotides forms a        plurality of tertiary structures, wherein the plurality of        tertiary structures comprises the first tertiary structure and        the secondary tertiary structure.    -   24. The composition of clause 23, wherein an axis of symmetry of        the first tertiary structure and an axis of symmetry of the        second tertiary structure are coplanar.    -   25. The composition of clause 23, wherein the axis of symmetry        of the first tertiary structure and the axis of symmetry of the        second tertiary structure are non-coplanar.    -   26. The composition of clause 23, wherein the axis of symmetry        of the first tertiary structure and the axis of symmetry of the        second tertiary structure are intersecting.    -   27. The composition of clause 23, wherein the axis of symmetry        of the first tertiary structure and the axis of symmetry of the        second tertiary structure are non-intersecting    -   28. The composition of any one of clauses 23-27, wherein the        plurality of tertiary structures surrounds an internal volume        region of the structured nucleic acid particle.    -   29. The composition of clause 28, wherein the internal volume        region comprises the display face or the capture face.    -   30. The composition of any one of the preceding clauses, further        comprising the analyte.    -   31. The composition of clause 30, wherein the display moiety is        coupled to the analyte.    -   32. The composition of any one of the preceding clauses, further        comprising the surface.    -   33. The composition of clause 32, wherein the capture moiety is        coupled to the surface.    -   34. The composition of any one clauses 30-33, wherein the        analyte comprises a biomolecule selected from the group        consisting of polypeptide, polysaccharide, nucleic acid, lipid,        and a combination thereof    -   35. The composition of any one of clauses 1-33, wherein the        analyte comprises a non-biological particle selected from the        group consisting of polymer, metal, metal oxide, ceramic,        semiconductor, mineral, and a combination thereof.    -   36. The composition of any one of the preceding clauses, further        comprising a second multifunctional moiety comprising a third        functional group and a fourth functional group.    -   37. The composition of clause 36, wherein the display moiety        comprises the third functional group and the capture moiety        comprises the fourth functional group.    -   38. The composition of clause 36, wherein the display moiety        does not comprise the third functional group or the fourth        functional group.    -   39. The composition of clause 36 or 37, wherein the fourth        functional group is configured to be coupled to the surface.    -   40. The composition of clause 39, wherein the fourth functional        group is coupled to the surface.    -   41. The composition of any one of clause 36-40, wherein the        third functional group is configured to be coupled to a second        analyte.    -   42. The composition of clause 41, wherein the third functional        group is coupled to the second analyte.    -   43. The composition of any one of clause 36-40, wherein the        third functional group is configured to be coupled to the        analyte.    -   44. The composition of clause 43, wherein the third functional        group is coupled to the analyte.    -   45. The composition of any one of clause 36-40, wherein the        third functional group is configured to be coupled to a        functional nucleic acid strand.    -   46. The composition of clause 45, wherein the functional nucleic        acid strand comprises a hybridization sequence, a priming        sequence, or a nucleic acid barcode.    -   47. The composition of clause 45 or 46, wherein the third        functional group is coupled to the functional nucleic acid        strand.    -   48. The composition of any one of clauses 32-47, wherein the        surface comprises a surface functional group that is configured        to couple to the second functional group.    -   49. The composition of clause 48, wherein the surface functional        group and the second functional group form a covalent bond.    -   50. The composition of clause 49, wherein the covalent bond is        formed by a click reaction.    -   51. The composition of any one of the preceding clauses, wherein        the structured nucleic acid particle comprises one or more        photocleavable linkers.    -   52. The composition of clause 51, wherein the multifunctional        moiety does not comprise a photocleavable linker.    -   53. The composition of any one of the preceding clauses, wherein        the structured nucleic acid particle comprises one or more        restriction sites.    -   54. The composition of clause 53, wherein the multifunctional        moiety does not comprise a restriction site of the one or more        restriction sites.    -   55. The composition of any one of the preceding clauses, wherein        the multifunctional moiety comprises a linker.    -   56. The composition of clause 55, wherein the linker comprises a        modified nucleotide.    -   57. The composition of clause 55 or 56, wherein the linker        comprises a linking moiety that is configured to couple one or        more additional molecules to the multifunctional moiety.    -   58. The composition of clause 57, wherein the one or more        additional molecules comprises a third multifunctional moiety,        wherein the third multifunctional moiety comprises a fifth        functional group and a sixth functional group.    -   59. The composition of clause 58, wherein the sixth functional        group is coupled to the linking moiety.    -   60. The composition of clause 58 or 59, wherein the third        multifunctional moiety is hybridized to the structured nucleic        acid particle, wherein the capture moiety comprises the fifth        functional group.    -   61. The composition of any one of clauses 58-60, wherein the        fifth functional group is configured to be coupled to the        surface.    -   62. The composition of clause 61, wherein the fifth functional        group is coupled to the surface.    -   63. The composition of any one of the preceding clauses, wherein        the capture moiety comprises a modifying moiety, selected from        the group consisting of an electrically-charged moiety, a        magnetic moiety, a steric moiety, an amphipathic moiety, a        hydrophobic moiety, and a hydrophilic moiety.    -   64. The composition of clause 63, wherein the        electrically-charged moiety comprises a single-stranded nucleic        acid.    -   65. The composition of clause 64, wherein the capture moiety        comprises a plurality of single-stranded nucleic acids.    -   66. The composition of any one of the preceding clauses, further        comprising a separating group, wherein the separating group is        configured to couple the analyte to the display moiety, thereby        creating a separation gap between the analyte and the structured        nucleic acid particle.    -   67. The composition of clause 66, wherein the separating group        comprises a rigid separating group selected from the group        comprising a polymer linker, a nucleic acid linker, and a        nanoparticle linker.    -   68. The composition of clause 67, wherein the nucleic acid        linker comprises a tertiary structure.    -   69. The composition of clause 67 or 68, wherein the separating        group comprises a flexible linker.    -   70. The composition of any one of clauses 66 to 69, wherein the        separation gap comprises a gap between the analyte and the        capture moiety.    -   71. The composition of any one of clauses 66-70, wherein the        separation gap comprises a gap between the analyte and a nearest        point of the structured nucleic acid particle.    -   72. The composition of any one of clauses 66-71, wherein the        separation gap is at least about 5 nanometers.    -   73. The composition of clause 72, wherein the separation gap is        no more than about 100 nanometers.    -   74. The composition of any one of clauses 22-74, wherein the        structured nucleic acid particle comprises two or more scaffold        strands.    -   75. The composition of clause 74, wherein an oligonucleotide of        a plurality of oligonucleotides hybridizes to at least two        scaffold strands of the two or more scaffold strands.    -   76. The composition of clause 75, wherein at least 10% of the        plurality of oligonucleotides hybridize to at least two scaffold        strands of the two or more scaffold strands.    -   77. The composition of any one of the preceding clauses, wherein        the multifunctional moiety is covalently cross-linked to the        structured nucleic acid particle.    -   78. The composition of any one of clauses 2-77, wherein the        nucleic acid strand hybridizes to the structured nucleic acid        particle with a characteristic melting temperature of at least        70 degrees Celsius (° C.).    -   79. A composition, comprising:        -   a structured nucleic acid particle (SNAP); and        -   a multifunctional moiety;        -   wherein the multifunctional moiety is coupled to the SNAP,            and wherein the multifunctional moiety is configured to form            a continuous linker from a surface to an analyte.    -   80. The composition of clause 79, wherein the multifunctional        moiety comprises a first functional group and a second        functional group.    -   81. The composition of clause 79 or 80, wherein the        multifunctional moiety comprises a nucleic acid strand that is        configured to couple to the SNAP.    -   82. The composition of clause 79 or 80, wherein the        multifunctional moiety does not comprise a nucleic acid.    -   83. The composition of clause 82, wherein the multifunctional        moiety further comprises a third functional group that is        configured to couple to the SNAP.    -   84. The composition of clause 83, wherein the third functional        group is configured to form a covalent bond with a complementary        functional group of the SNAP.    -   85. The composition of clause 83, wherein the third functional        group is configured to non-covalently couple to the SNAP.    -   86. The composition of any one of clauses 80-85, wherein the        first functional group is configured to couple to the surface,        and the second functional group is configured to couple to the        analyte.    -   87. The composition of any one of clauses 79-86, wherein the        multifunctional moiety is coupled to the SNAP.    -   88. A structured nucleic acid particle (SNAP) complex,        comprising two or more SNAPs, wherein each SNAP of the two or        more SNAPs is selected independently from the group consisting        of a display SNAP, a utility SNAP, or a combination thereof;        -   wherein the display SNAP comprises a display moiety that is            configured to couple to an analyte;        -   wherein the utility SNAP comprises a capture moiety that is            configured to couple with a surface; and        -   wherein the two or more SNAPs are coupled to form the SNAP            complex.    -   89. The SNAP complex of clause 88, wherein the utility SNAP        comprises a capture SNAP, a coupling SNAP, a structural SNAP, or        a combination thereof    -   90. The SNAP complex of clause 89, wherein the SNAP complex        comprises a display SNAP and a capture SNAP.    -   91. The SNAP complex of clause 90, wherein the display SNAP or        the capture SNAP comprises a DNA nanoball or a DNA origami.    -   92. The SNAP complex of clause 91, wherein the DNA origami        comprises a scaffold nucleic acid strand and a plurality of        oligonucleotides that are coupled to the scaffold nucleic acid        strand.    -   93. The SNAP complex of clause 92, wherein the scaffold strand        comprises a circular strand or a non-circular strand having a        length of at least 1000 nucleotides.    -   94. The SNAP complex of clause 92 or 93, wherein an        oligonucleotide of the plurality of oligonucleotides comprises        the capture moiety.    -   95. The SNAP complex of any one of clauses 88 to 94, wherein the        capture moiety is selected from the moiety consisting of a        reactive moiety, an electrically-charged moiety, a magnetic        moiety, streptavidin, and biotin.    -   96. The SNAP complex of clause 95, wherein the reactive moiety        comprises a click reaction reagent.    -   97. The SNAP complex of any one of clauses 92-96, wherein the        oligonucleotide of the plurality of oligonucleotides further        comprises the display moiety.    -   98. The SNAP complex of clause 92-97, wherein an oligonucleotide        of the plurality of oligonucleotides comprises the capture        moiety.    -   99. The SNAP complex of any one of clauses 88-98, wherein the        capture moiety is selected from the moiety consisting of a        reactive moiety, an electrically-charged moiety, a magnetic        moiety, streptavidin, and biotin.    -   100. The SNAP complex of clause 99, wherein the reactive moiety        comprises a click reaction reagent.    -   101. The SNAP complex of any one of clauses 88-100, wherein the        display moiety is attached to a face of the display SNAP that is        offset from a face of the display SNAP to which the capture        moiety is attached by an angle of about 180°.    -   102. The SNAP complex of any one of clauses 88-101, wherein the        display moiety is attached to a face of the display SNAP that is        offset from a face of the SNAP to which the capture moiety is        attached by an angle of less than about 180°.    -   103. The SNAP complex of any one of clauses 90-102, wherein the        display SNAP comprises a utility face, wherein the utility face        comprises a capture moiety, a detectable label, or a sterically        blocking moiety.    -   104. The SNAP complex of clause 103, wherein the detectable        label comprises a fluorescent label, a luminescent label, a        nucleic acid barcode, an isotope, or a radiolabel.    -   105. The SNAP complex of any one of clauses 90-104, wherein the        display SNAP comprises a first SNAP coupling moiety and the        capture SNAP comprises a second SNAP coupling moiety, wherein        the display SNAP is coupled to the capture SNAP by coupling of        the first SNAP coupling moiety to the second SNAP coupling        moiety.    -   106. The SNAP complex of clause 105, wherein the first SNAP        coupling moiety and the second SNAP coupling moiety form a        covalent bond.    -   107. The SNAP complex of clause 105 or 106, wherein the first        SNAP coupling moiety and the second SNAP coupling moiety        comprise a complementary pair of click reaction reagents.    -   108. The SNAP complex of clause 105, wherein the first SNAP        coupling moiety and the second SNAP coupling moiety form a        non-covalent bond.    -   109. The SNAP complex of clause 108, wherein the non-covalent        bond comprises a hydrogen bond, a nucleic acid base pair bond,        or a streptavidin-biotin bond.    -   110. The SNAP complex of any one of clauses 88-109, wherein the        SNAP complex comprises a plurality of capture SNAPs and a single        display SNAP.    -   111. The SNAP complex of clause 110, wherein the plurality of        SNAPs comprises at least about 4 capture SNAPs.    -   112. The SNAP complex of any one of clauses 88-111, wherein the        SNAP complex comprises a ratio of more than one capture SNAP per        display SNAP.    -   113. The SNAP complex of clause 112, wherein the SNAP complex        comprises at least two capture SNAPs per display SNAP.    -   114. The SNAP complex of clause 113, wherein the SNAP complex        comprises at least four capture SNAPs per display SNAP.    -   115. The SNAP complex of any one of clauses 88-114, wherein the        SNAP complex comprises a display SNAP and two or more capture        SNAPs coupled to one or more faces of the display SNAP.    -   116. The SNAP complex of clause 115, wherein a first capture        SNAP of the two or more capture SNAPs is coupled to a first face        of the display SNAP, and wherein a second capture SNAP of the        two or more capture SNAPs is coupled to a second face of the        display SNAP.    -   117. The SNAP complex of clause 116, wherein a face of the first        capture SNAP is coupled to a face of the second capture SNAP.    -   118. The SNAP complex of clause 116, wherein the first capture        SNAP is not coupled to the second capture SNAP.    -   119. The SNAP complex of any one of clauses 116-118, wherein the        SNAP complex further comprises a third capture SNAP.    -   120. The SNAP complex of clause 119, wherein the third capture        SNAP is coupled to a third face of the display SNAP.    -   121. The SNAP complex of clause 119 or 120, wherein the third        capture SNAP is coupled to a face of the first capture SNAP, a        face of the second capture SNAP, or a combination thereof.    -   122. The SNAP complex of any one of clauses 119-121, wherein the        face of the first capture SNAP is larger than the first face of        the display SNAP.    -   123. The SNAP complex of any one of clauses 119-121, wherein the        face of the first capture SNAP is about the same size as the        first face of the display SNAP.    -   124. The SNAP complex of any one of clauses 119-121, wherein the        face of the first capture SNAP is smaller than the first face of        the display SNAP.    -   125. The SNAP complex of any one of clauses 119-124, wherein the        face of the second capture SNAP is larger than the first face of        the display SNAP.    -   126. The SNAP complex of any one of clauses 119-124, wherein the        face of the second capture SNAP is about the same size as the        first face of the display SNAP.    -   127. The SNAP complex of any one of clauses 119-124, wherein the        face of the second capture SNAP is smaller than the first face        of the display SNAP.    -   128. The SNAP complex of any one of clauses 115-127, wherein the        one or more faces of the display SNAP do not comprise the        capture moiety.    -   129. The SNAP complex of any one of clauses 115-127, wherein the        one or more faces of the display SNAP comprise at least about        two faces.    -   130. The SNAP complex of clause 129, wherein the one or more        faces of the display SNAP comprise at least about four faces.    -   131. The SNAP complex of any one of clauses 128-130, wherein        each face of the one or more faces is coupled to a capture SNAP.    -   132. The SNAP complex of any one of clauses 128-130, wherein at        least one face of the one or more faces is not coupled to a        capture SNAP.    -   133. The SNAP complex of any one of clauses 88-132, wherein the        SNAP complex comprises at least one axis of symmetry.    -   134. The method of clause 133, wherein the axis of symmetry        comprises a rotational axis of symmetry or a reflection axis of        symmetry.    -   135. The method of clause 134, wherein the SNAP complex        comprises a rotational axis of symmetry and a reflection axis of        symmetry.    -   136. The SNAP complex of any clauses of clauses 88-132, wherein        the SNAP complex has no axis of symmetry.    -   137. The SNAP complex of any one of clauses 88-136, wherein the        SNAP complex has a square, rectangular, triangular, cross, or        polygonal conformation.    -   138. The SNAP complex of any one of clauses 89-137, wherein the        capture SNAP comprises a utility face containing a sterically        blocking moiety or a SNAP complex coupling moiety.    -   139. The SNAP complex of clause 138, wherein the sterically        blocking moiety is selected from the moiety consisting of        polyethylene glycol (PEG), polyethylene oxide (PEO), or        dextrans.    -   140. The SNAP complex of clause 138, wherein the SNAP complex        coupling moiety is configured to couple the SNAP complex to a        second SNAP complex.    -   141. The SNAP complex of clause 138 or 140, wherein the complex        coupling moiety is configured to form a covalent bond or a        non-covalent bond.    -   142. The SNAP complex of any one of clauses 89-141, wherein the        display SNAP comprises a capture moiety.    -   143. The SNAP complex of clause 142, wherein the capture moiety        of the display SNAP or the capture moiety of the capture SNAP        comprises a modifying moiety selected from the moiety consisting        of an electrically-charged moiety, a magnetic moiety, a steric        moiety, an amphipathic moiety, a hydrophobic moiety, and a        hydrophilic moiety.    -   144. The SNAP complex of any clause 142 or 143, wherein the        capture moiety of the display SNAP is different from the capture        moiety of the capture SNAP.    -   145. The SNAP complex of clause 142 or 143, wherein the capture        moiety of the display SNAP is the same as the capture moiety of        the capture SNAP.    -   146. The SNAP complex of any one of clauses 88-145, wherein the        analyte is coupled to the display SNAP.    -   147. The SNAP complex of clause 146, wherein a single analyte is        coupled to the display SNAP.    -   148. The SNAP complex of clause 146, wherein a plurality of        polypeptides is coupled to the display SNAP.    -   149. The SNAP complex of any one of clauses 88-148, wherein the        SNAP complex comprises a plurality of display SNAPs.    -   150. The SNAP complex of clause 149, wherein a display SNAP of        the plurality of display SNAPs is coupled to the analyte.    -   151. The SNAP complex of any one of clauses 88-150, wherein a        first SNAP comprising a first capture face of the two or more        SNAPs and a second SNAP comprising a second capture face of the        two or more SNAPs are coupled rigidly.    -   152. The SNAP complex of clause 151, wherein the capture face of        the first SNAP and the capture face of the second SNAP are        substantially coplanar.    -   153. The SNAP complex of clause 151, wherein the capture face of        the first SNAP and the capture face of the second SNAP are not        coplanar.    -   154. The SNAP complex of clause 153, wherein the capture face of        the first SNAP is oriented at an angle of at least about 10°        relative to the capture face of the second SNAP.    -   155. The SNAP complex of any one of clauses 88-154, wherein the        SNAP complex comprises one or more structural SNAPs.    -   156. The SNAP complex of clause 155, wherein the one or more        structural SNAPs comprise a separating SNAP, a supporting SNAP,        or a modifying SNAP.    -   157. The SNAP complex of clause 156, wherein the separating SNAP        is configured to form a separation gap between the analyte and        the surface.    -   158. The SNAP complex of clause 157, wherein the separation gap        is at least about 5 nm.    -   159. The SNAP complex of clause 157 or 158, wherein the        separation gap is no more than about 100 nm.    -   160. The SNAP complex of clause 156, wherein the supporting SNAP        or the modifying SNAP is coupled at least one SNAP of the two or        more SNAPs.    -   161. A structured nucleic acid particle (SNAP) composition,        comprising:        -   a material comprising a surface; and        -   two or more SNAPs, wherein each SNAP of the two or more            SNAPs is selected independently from the group consisting of            a display SNAP, a utility SNAP, or a combination thereof;        -   wherein the display SNAP comprises a display moiety that is            configured to couple to an analyte,        -   wherein the two or more SNAPs are coupled to the surface;            and        -   wherein a first SNAP of the two or more SNAPs is coupled to            a second SNAP of the two or more SNAPs, thereby forming a            SNAP complex.    -   162. The composition of clause 161, wherein the utility SNAP        comprises a capture SNAP, a coupling SNAP, a structural SNAP, or        a combination thereof.    -   163. The composition of clause 161 or 162, wherein the material        comprises a solid support.    -   164. The composition of any one of clauses 161-163, wherein the        material comprises silicon, fused silica, quartz, mica, or        glass.    -   165. The composition of clause 163 or 164, wherein the surface        comprises a layer selected from the group consisting of a metal,        a metal oxide, or a polymer.    -   166. The composition of any one of clauses 161-165, wherein the        surface further comprises a functional group that is coupled to        a first SNAP of the two or more SNAPs.    -   167. The composition of clause 166, wherein the first SNAP of        the two or more SNAPs comprises a capture moiety that is coupled        to the functional group of the material.    -   168. The composition of 166 or 167, wherein the functional group        is coupled to a display SNAP or a capture SNAP.    -   169. The composition of any one of clauses 166-168, wherein the        functional group is configured to form an electrostatic,        magnetic, covalent, or non-covalent interaction with the SNAP        complex.    -   170. The composition of clause 161, wherein the first SNAP of        the two or more SNAPs comprises a capture moiety that is        directly coupled to the material.    -   171. The composition of clause 170, wherein the material        comprises a metal oxide.    -   172. The composition of any one of clauses 161-171, wherein the        surface is patterned with a plurality of binding sites separated        by interstitial regions, wherein each binding site is configured        to bind the SNAP complex, wherein the interstitial regions are        configured to not bind the SNAP complex.    -   173. The composition of clause 161 or 162, wherein the surface        comprises a phase boundary between two fluids.    -   174. The composition of clause 173, wherein the phase boundary        comprises a gas/liquid interface or a liquid/liquid interface.    -   175. The composition of clause any one of clauses 161-174,        wherein the analyte is coupled to the SNAP complex.    -   176. The composition of any one of clauses 161 to 175, wherein        the SNAP complex comprises an effective surface area of at least        5000 square nanometers (nm²).    -   177. The composition of clause 176, wherein the SNAP complex        comprises an effective surface area of at least 10000 nm².    -   178. The composition of clause 177, wherein the SNAP complex        comprises an effective surface area of at least 100000 nm².    -   179. The composition of any one of clauses 161 to 178, wherein        the effective surface area of the SNAP complex comprises at        least 25% of the effective surface area of a binding site of the        material that is configured to couple to the SNAP complex.    -   180. The composition of clause 179, wherein the effective        surface area of the SNAP complex comprises at least 50% of the        effective surface area of a binding site of the material that is        configured to couple to the SNAP complex.    -   181. The composition of any one of clauses 179 or 180, wherein        the conformation of the SNAP complex coupled to the binding site        prevents a second SNAP complex from coupling to the binding        site.    -   182. The SNAP complex of any one of clauses 161-181, wherein the        SNAP complex has a square, rectangular, triangular, cross, or        polygonal conformation.    -   183. The SNAP complex of any one of clauses 161-182, wherein the        surface comprises a binding structure that conforms to the        conformation of the SNAP complex.    -   184. The SNAP complex of clause 183, wherein the binding        structure comprises a two-dimensional or three-dimensional        geometry.    -   185. The SNAP complex of clause 183 or 184, wherein the surface        is patterned with a plurality of binding sites separated by        interstitial regions, wherein each binding site comprises the        binding structure, wherein each binding structure is configured        to bind the SNAP complex, wherein the interstitial regions are        configured to not bind the SNAP complex.    -   186. A structured nucleic acid particle (SNAP) composition,        comprising:        -   an analyte;        -   a display SNAP; and        -   one or more SNAPs selected from the group consisting of a            display SNAP, a utility SNAP, and combinations thereof;        -   wherein the display SNAP comprises a display moiety that is            configured to couple to the analyte;        -   wherein the display SNAP is coupled to the analyte; and        -   wherein the display SNAP is coupled to the one or more            SNAPs, thereby forming a SNAP complex.    -   187. A structured nucleic acid particle (SNAP) composition,        comprising:        -   a material comprising a surface;        -   an analyte;        -   a display SNAP; and        -   one or more SNAPs selected from the group consisting of a            display SNAP, a utility SNAP, and combinations thereof;        -   wherein the display SNAP comprises a display moiety that is            configured to couple to the analyte;        -   wherein the display SNAP is coupled to the analyte;        -   wherein the display SNAP is coupled to the one or more            SNAPs, thereby forming a SNAP complex; and        -   wherein the SNAP complex is coupled to the surface.    -   188. An array, comprising:        -   a plurality of SNAP complexes; and        -   a material comprising a surface;        -   wherein each of the SNAP complexes is coupled to the            surface; and        -   wherein each SNAP complex of the plurality of SNAP complexes            is coupled to one or more other SNAP complexes of the            plurality of SNAP complexes;        -   wherein each SNAP complex of the plurality of SNAP complexes            comprises two or more SNAPs selected independently from the            group consisting of a display SNAP, a utility SNAP, and            combinations thereof    -   189. The array of clause 188, wherein the utility SNAP comprises        a capture SNAP, a coupling SNAP, a structural SNAP, or a        combination thereof.    -   190. The array of clause 188 or 189, wherein each SNAP complex        of the plurality of SNAP complexes is reversibly coupled to one        or more other SNAP complexes.    -   191. The array of clause 190, wherein a first SNAP complex of        the plurality of SNAP complexes remains reversibly coupled to a        second SNAP complex of the plurality of SNAP complexes for at        least about 1 day.    -   192. The array clause 188 or 189, wherein each SNAP complex of        the plurality of SNAP complexes is irreversibly coupled to one        or more other SNAP complexes.    -   193. The array of any one of clauses 188-192, wherein each        display SNAP of the array comprises a display moiety.    -   194. The array of clause 193, wherein each display moiety is        separated from an adjacent display moiety by a distance of at        least about 50 nanometers (nm).    -   195. The array of clause 194, wherein each display moiety is        separated from an adjacent display moiety by a distance of at        least about 100 nm.    -   196. The array of clause 195, wherein each display moiety is        separated from an adjacent display moiety by a distance of at        least about 300 nm.    -   197. The array of any one of clauses 188-196, wherein the        surface is patterned with a plurality of binding sites separated        by interstitial regions, wherein each binding site is configured        to bind a plurality of SNAP complexes, wherein the interstitial        regions are configured to not bind the SNAP complex.    -   198. The array of clause 197, wherein each binding site is        configured to bind to two or more coupled SNAP complexes.    -   199. The array of any one of clauses 188-198, wherein a        plurality of SNAP complexes is coupled to a plurality of        analytes.    -   200. The array of any one of clauses 188-199, wherein the array        comprises two or more species of SNAP complexes, wherein each        species of the two or more species of SNAP complexes is        chemically or conformationally distinct.    -   201. The array of clause 200, wherein a plurality of a first        species of SNAP complexes is segregated from a plurality of a        second species of SNAP complexes.    -   202. The array of clause 200 or 201, wherein the array comprises        a homogeneous or heterogeneous mixture of the two or more        species of SNAP complexes.    -   203. The method of any one of clauses 200-202, wherein each        species of the two or more species of SNAP complexes is        configured to be coupled to a single species of analyte of a        plurality of species of analytes.    -   204. The array of clause 203, wherein the single species of        analyte is selected from the group comprising sample analytes,        control analytes, standard analytes, and inert analytes.    -   205. A method of forming an array, comprising:        -   providing a plurality of SNAP complexes;        -   coupling each SNAP complex of the plurality of SNAP            complexes to one or more additional SNAP complexes from the            plurality of SNAP complexes; and        -   coupling each SNAP complex of the plurality of SNAP            complexes with a surface;        -   wherein each SNAP complex comprises a display SNAP and one            or more utility SNAPs, and wherein each SNAP complex            comprises a coupling moiety that couples with the surface,            thereby forming an array.    -   206. The method of clause 205, wherein the utility SNAP        comprises a capture SNAP, a coupling SNAP, a structural SNAP, or        a combination thereof.    -   207. The method of clause 205 or 206, wherein the associating        each SNAP complex of the plurality of SNAP complexes occurs        before the coupling each SNAP complex of the plurality of SNAP        complexes to one or more additional SNAP complexes.    -   208. The method of clause 205 or 206, wherein the associating        each SNAP complex of the plurality of SNAP complexes occurs        after the coupling each SNAP complex of the plurality of SNAP        complexes to one or more additional SNAP complexes.    -   209. The method of any one of clauses 205-207, wherein the        display SNAP comprises a display moiety.    -   210. The method of clause 209, further comprising a step of        coupling an analyte to the display moiety.    -   211. The method of clause 210, wherein the analyte is coupled to        the display moiety after the coupling of each SNAP complex of        the plurality of SNAP complexes with the surface.    -   212. The method of clause 210, wherein the analyte is coupled to        the display moiety before the coupling of each SNAP complex of        the plurality of SNAP complexes with the surface.    -   213. The method of clause 210, wherein the analyte is coupled to        the display moiety after the coupling of each SNAP complex of        the plurality of SNAP complexes to one or more additional SNAP        complexes from the plurality of SNAP complexes.    -   214. The method of clause 210, wherein the analyte is coupled to        the display moiety before the coupling of each SNAP complex of        the plurality of SNAP complexes to one or more additional SNAP        complexes from the plurality of SNAP complexes.    -   215. The method of clause 210, wherein the polypeptide is        coupled to the display moiety after the providing of the        plurality of SNAP complexes.    -   216. The method of clause 210, wherein the analyte is coupled to        the display moiety before the providing of the plurality of SNAP        complexes.    -   217. The method of any one of clauses 210-216, wherein the        analyte is covalently coupled to the display moiety.    -   218. The method of clause 217, wherein the analyte is covalently        coupled to the display moiety by a click reaction.    -   219. The method of clause 217 or 218, wherein the coupling        occurs in the presence of a surfactant.    -   220. A composition, comprising:        -   a. a structured nucleic acid particle, wherein the            structured nucleic acid particle comprises:            -   i. a retaining component;            -   ii. a display moiety comprising a coupling group that is                configured to couple an analyte, wherein the display                moiety is coupled to the retaining component; and            -   iii. a capture moiety that is configured to couple with                a surface, wherein the capture moiety comprises a                plurality of first surface-interacting oligonucleotides,                and wherein each first surface-interacting                oligonucleotide of the plurality of first                surface-interacting oligonucleotides comprises a first                nucleic acid strand that is coupled to the retaining                component and a first surface-interacting moiety,                wherein the first surface-interacting moiety is                configured to form a coupling interaction with a                surface-linked moiety;        -   wherein the capture moiety is restrained from contacting the            display moiety by the retaining component; and        -   b. an analyte comprising a complementary coupling group that            is configured to couple to the display moiety of the            structured nucleic acid particle.    -   221. The composition of clause 220, wherein the first        surface-interacting moiety comprises a second nucleic acid        strand.    -   222. The composition of clause 221, wherein the second nucleic        acid strand is configured to hybridize with a complementary        nucleic acid strand of the surface-linked moiety.    -   223. The composition of any one of clauses 220-222, wherein the        first surface-interacting moiety comprises a capture group        selected from the group consisting of a reactive group, an        electrically-charged group, a magnetic group, and a component of        a binding pair.    -   224. The composition of clause 223, wherein the binding pair is        selected from the group consisting of streptavidin-biotin,        SpyCatcher-Spytag, SnoopCatcher-Snooptag, and SdyCatcher-Sdytag.    -   225. The composition of anyone of clauses 220-224, wherein the        first surface-interacting moiety comprises a linker.    -   226. The composition of clause 225 wherein the linker comprises        a hydrophobic linker, a hydrophilic linker, or a cleavable        linker.    -   227. The composition of clause 223, wherein the reactive group        is configured to conjugate with the surface-linked moiety via a        Click-type reaction.    -   228. The composition of clause 223, wherein the first        surface-interacting moiety comprises a group that is configured        to form a non-covalent interaction selected from the group        consisting of an electrostatic interaction, a magnetic        interaction, a hydrogen bond, an ionic bond, a van der Waals        bond, a hydrophobic interaction, or a hydrophilic interaction.    -   229. The composition of clause 223 or 228, wherein the first        surface-interacting moiety comprises a nanoparticle selected        from the group consisting of an inorganic nanoparticle, a carbon        nanoparticle, a polymer nanoparticle, and a biopolymer.    -   230. The composition of any one of clauses 220-229, wherein the        structured nucleic acid particle comprises:        -   a. a scaffold nucleic acid strand; and        -   b. a plurality of staple nucleic acid strands, wherein each            staple nucleic acid strand is hybridized to non-contiguous            regions of the scaffold nucleic acid strand.    -   231. The composition of clause 230, wherein the plurality of        staple nucleic acid strands comprises a first        surface-interacting oligonucleotide of the plurality of first        surface-interacting oligonucleotides.    -   232. The composition of clause 231, wherein a coupling of the        first surface-interacting oligonucleotide forms a tertiary        structure of the structured nucleic acid particle.    -   233. The composition of clause 232, wherein the capture moiety        comprises the tertiary structure.    -   234. The composition of clause 232 or 233, wherein the display        moiety comprises the tertiary structure.    -   235. The composition of any one of clauses 220-234, wherein a        first surface-interacting oligonucleotide of the plurality of        first surface-interacting oligonucleotides comprises a first        nucleotide sequence that is configured to couple to the        structured nucleic acid particle and a second nucleotide        sequence that is configured to couple to a complementary        oligonucleotide of the surface-linked moiety.    -   236. The composition of clause 235, wherein the second        nucleotide sequence comprises a nucleotide sequence with no        self-complementarity of more than three contiguous nucleotides.    -   237. The composition of clause 235, wherein the second        nucleotide sequence comprises no more than 3 deoxyribonucleotide        species selected from the group consisting of deoxyadenosine,        deoxycytosine, deoxyguanosine, and deoxythymidine.    -   238. The composition of clause 235, wherein the second        nucleotide sequence comprises a nucleotide sequence with        self-complementarity of at least four contiguous nucleotides.    -   239. The composition of clause 238, wherein the        self-complementarity comprises a nucleic acid secondary        structure selected from the group consisting of a double-helix,        a stem loop, a pseudoknot, and a G-quadruplex.    -   240. The composition of any one of clauses 235-239, wherein the        first surface-interacting oligonucleotide of the plurality of        first surface-interacting oligonucleotides comprises a        homopolymer sequence of at least four nucleotides selected from        the group consisting of a poly-deoxyadenosine sequence, a        poly-deoxycytosine sequence, a poly-deoxyguanosine sequence, or        a poly-deoxythymidine sequence.    -   241. The composition of any one of clauses 235-240, wherein the        second nucleotide sequence comprises at least 5 nucleotides.    -   242. The composition of clause 241, wherein the second        nucleotide sequence comprises at least 10 nucleotides.    -   243. The composition of clause 242, wherein the second        nucleotide sequence comprises at least 15 nucleotides.    -   244. The composition of any one of clauses 241-243, wherein the        second nucleotide sequence comprises no more than 100        nucleotides.    -   245. The composition of any one of clauses 220-244, wherein the        first surface-interacting oligonucleotide of the plurality of        first surface-interacting oligonucleotides further comprises the        coupling group.    -   246. The composition of clause 245, wherein the first        surface-interacting oligonucleotide is coupled to the analyte.    -   247. The composition of any one of clauses 220-246, wherein the        plurality of first surface-interacting oligonucleotides        comprises at least 5 first surface-interacting oligonucleotides.    -   248. The composition of clause 247, wherein the plurality of        first surface-interacting oligonucleotides comprises at least 10        first surface-interacting oligonucleotides.    -   249. The composition of clause 248, the plurality of first        surface-interacting oligonucleotides comprises at least 20 first        surface-interacting oligonucleotides.    -   250. The composition of any one of clauses 247-249, wherein the        capture moiety comprises an average first surface-interacting        oligonucleotide density of at least 0.0001 single-stranded        oligonucleotides per square nanometer of effective surface area.    -   251. The composition of clause 250, wherein the capture moiety        comprises an average first surface-interacting oligonucleotide        density of at least 0.001 single-stranded oligonucleotides per        square nanometer of effective surface area.    -   252. The composition of clause 251, wherein the capture moiety        comprises an average first surface-interacting oligonucleotide        density of at least 0.01 single-stranded oligonucleotides per        square nanometer of effective surface area.    -   253. The composition of any one of clauses 247-252, wherein the        first surface-interacting oligonucleotide density is        substantially uniform over the effective surface area of the        capture moiety.    -   254. The composition of any one of clauses 247-252, wherein the        first surface-interacting oligonucleotide density is not        substantially uniform over the effective surface area of the        capture moiety.    -   255. The composition of clause 254, wherein a fraction of the        plurality of first surface-interacting oligonucleotides is        located near a central region of the capture moiety.    -   256. The composition of clause 254 or 255, wherein a fraction of        the plurality of first surface-interacting oligonucleotides is        concentrated near an outer region of the capture moiety.    -   257. The composition of any one of clauses 220-256, wherein the        capture moiety further comprises a second surface-interacting        oligonucleotide, wherein the second surface-interacting        oligonucleotide comprises a first nucleotide sequence that is        configured to couple to the structured nucleic acid particle and        a second surface-interacting moiety, wherein the second        surface-interacting moiety of the second surface-interacting        oligonucleotide differs from the first surface-interacting        moiety of the first surface-interacting oligonucleotide of the        plurality of first surface-interacting oligonucleotides.    -   258. The composition of clause 257, wherein the first        surface-interacting moiety comprises a nucleic acid with a first        nucleic acid sequence and the second surface-interacting moiety        comprises a nucleic acid with a second nucleic acid sequence,        wherein the first nucleic acid sequence differs from the second        nucleic acid sequence.    -   259. The composition of clause 257, wherein the first        surface-interacting moiety comprises a nucleic acid with a first        nucleic acid sequence and the second surface-interacting moiety        comprises a reactive group that is configured to form a covalent        bond with a coupling surface or a non-nucleic acid group that is        configured to form a non-covalent interaction with a coupling        surface.    -   260. A composition, comprising:        -   a. a structured nucleic acid particle, wherein the            structured nucleic acid particle comprises:            -   i. a retaining component;            -   ii. a display moiety that is coupled to the retaining                component; and            -   iii. a capture moiety that is coupled to the retaining                component, wherein the capture moiety comprises a                plurality of oligonucleotides, and wherein each                oligonucleotide of the plurality of oligonucleotides                comprises a surface-interacting moiety; and        -   b. a solid support comprising a coupling surface, wherein            the surface comprises a surface-linked moiety, and wherein a            surface-interacting moiety of the plurality of            surface-interacting moieties is coupled to the            surface-linked, wherein the display moiety is restrained            from contacting the surface by the retaining component.    -   261. The composition of clause 260, further comprising an        analyte coupled to the display moiety.    -   262. The composition of clause 261, wherein the analyte is        restrained from contacting the surface by the retaining        component.    -   263. The composition of clause 260, wherein the solid support        comprises an address comprising the one or more surface-linked        moieties, wherein the address is resolvable at single-analyte        resolution.    -   264. The composition of clause 261, wherein the address        comprises one or more surfaces, wherein the one or more surfaces        comprises the coupling surface, and wherein the coupling surface        comprises the one or more surface-linked moieties.    -   265. The composition of clause 262, wherein the one or more        surfaces form a three-dimensional structure of the solid        support.    -   266. The composition of clause 263, wherein the        three-dimensional structure comprises a raised structure or a        well structure.    -   267. The composition of any one of clauses 260-264, wherein the        coupling of the structured nucleic acid particle to the solid        support occludes the display moiety from contacting the coupling        surface.    -   268. The composition of any one of clauses 260-265, wherein the        coupling surface comprises a surface area that is larger than        the effective surface area of the capture moiety of the        structured nucleic acid particle.    -   269. The composition of any one of clauses 260-265, wherein the        coupling surface comprises a surface area that is smaller than        the effective surface area of the capture moiety of the        structured nucleic acid particle.    -   270. The composition of any one of clauses 260-267, wherein the        one or more surface-linked moieties comprises one or more        complementary oligonucleotides, wherein a complementary        oligonucleotide of the plurality of complementary        oligonucleotides is configured to couple to the        surface-interacting moiety, and wherein the surface-interacting        moiety comprises a nucleic acid strand with a nucleotide        sequence that is configured to hybridize with the complementary        oligonucleotide.    -   271. The composition of any one of clauses 260-268, wherein the        one or more surface-linked moieties comprises one or more        complementary reactive groups, wherein a complementary reactive        group of the one or more complementary reactive groups is        configured to couple to the surface-interacting moiety, and        wherein the surface-interacting moiety comprises a reactive        groups that is configured to couple to the complementary        reactive group.    -   272. The composition of any one of clauses 260-269, wherein the        one or more surface-linked moieties comprises one or more        surface groups, wherein a surface group of the one or more        complementary reactive groups is configured to form a coupling        interaction with the surface-interacting moiety, and wherein the        coupling interaction comprises an electrostatic interaction, a        magnetic interaction, a hydrogen bond, an ionic bond, a van der        Waals bond, a hydrophobic interaction, or a hydrophilic        interaction.    -   273. The composition of any one of clauses 260-270, wherein the        coupling surface comprises a plurality of surface-linked        moieties.    -   274. The composition of clause 271, wherein the surface-linked        moiety density of the coupling surface is substantially uniform        over the coupling surface.    -   275. The composition of clause 271, wherein the surface-linked        moiety density of the coupling surface is not substantially        uniform over the coupling surface.    -   276. The composition of clause 273, wherein a fraction of the        plurality of surface-linked moieties is located within a central        region of the coupling surface.    -   277. The composition of clause 273 or 274, wherein a second        fraction of the plurality of surface-linked moieties is located        within an outer region of the coupling surface.    -   278. The composition of any one of clauses 271-275, wherein a        fraction of surface-interacting moieties of the plurality of        oligonucleotides is coupled to a fraction of surface-linked        moieties of the plurality of surface-linked moieties.    -   279. The composition of clause 276, wherein the fraction of        surface-interacting moieties comprises at least 0.1.    -   280. The composition of clause 277, wherein the fraction of        surface-interacting moieties comprises at least 0.5.    -   281. The composition of clause 277 or 278, wherein the fraction        of surface-interacting moieties comprises less than 1.0.    -   282. The composition of any one of clauses 277-279, wherein the        fraction of surface-linked moieties comprises at least 0.01.    -   283. The composition of clause 280, wherein the fraction of        surface-linked moieties comprises at least 0.1.    -   284. The composition of clause 281, wherein the fraction of        surface-linked moieties comprises less than 1.0.    -   285. The composition of any one of clauses 260-284, wherein the        solid support further comprises a passivating layer.    -   286. The composition of clause 285, wherein the passivating        layer comprises a plurality of molecules that are configured to        prevent non-specific binding of a molecule to the solid support.    -   287. The composition of clause 286, wherein the plurality of        molecules comprises a plurality of surface-linked molecular        chains selected from the groups consisting of polyethylene        glycol, polyethylene oxide, an alkane, a nucleic acid, or a        dextran.    -   288. The composition of clause 286 or 287, wherein each molecule        of the plurality of molecules comprises a surface-linked moiety        of the one or more surface-linked moieties.    -   289. The composition of any one of clauses 286-288, wherein each        molecule of the plurality of molecules further comprises a        linking group that couples a surface-linked moiety of the one or        more surface-linked moieties to the coupling surface.    -   290. The composition of clause 289, wherein the linking group        comprises a silane, a phosphate, or a phosphonate.    -   291. A method of identifying a polypeptide, the method        comprising:        -   a. providing a composition of any one of clauses 260-290,            wherein the polypeptide is coupled to the display moiety;        -   b. contacting the solid support with a plurality of            detectable affinity reagents;        -   c. detecting presence or absence of binding of the            detectable affinity reagent of the plurality of detectable            affinity agents to the polypeptide;        -   d. optionally repeating steps b)-c) with a second plurality            of detectable affinity reagents; and        -   e. based upon the presence or absences of binding of one or            more of the affinity reagents, identifying the polypeptide.    -   292. The method of clause 291, wherein the detecting presence or        absence of binding comprises detecting a signal from the        detectable affinity reagent of the plurality of detectable        affinity reagents.    -   293. The method of clause 292, wherein the detectable signal        comprises fluorescence, luminescence, luminescence lifetime, or        signal encoding.    -   294. The method of clause 293, wherein the signal encoding        comprises transferring a nucleic acid barcode or a peptide        barcode from the detectable affinity reagent to a recording        nucleic acid or peptide.    -   295. A method of sequencing a polypeptide, the method        comprising:        -   a. providing a composition of any one of clauses 260-290,            wherein the polypeptide is coupled to the display moiety;        -   b. removing a terminal amino acid residue of the polypeptide            by an Edman-type degradation reaction;        -   c. identifying the terminal amino acid residue; and        -   d. repeating steps b-c) until a sequence of amino acid            residues has been identified for the polypeptide.    -   296. The method of clause 295, wherein the identifying the        terminal amino acid residue comprises:        -   a. contacting the polypeptide with an affinity reagent            comprising a binding specificity for the terminal amino acid            residue; and        -   b. detecting presence or absence of the affinity reagent,            wherein the affinity reagent is configured to produce a            distinguishable signal corresponding to the terminal amino            acid residue, wherein the distinguishable signal is            detectable by fluorescence, luminescence, or luminescence            lifetime.    -   297. The method of clause 296, wherein the distinguishable        signal is detectable by fluorescence, luminescence, or        luminescence lifetime.    -   298. The method of clause 296, wherein the identifying the        terminal amino acid residue comprises performing a        fluorosequencing reaction on the polypeptide.    -   299. A single-analyte array, comprising:        -   a. a solid support comprising a plurality of addresses,            wherein each address of the plurality of addresses is            resolvable at single-analyte resolution, wherein each            address comprises a coupling surface, and wherein each            coupling surface comprises one or more surface-linked            moieties;        -   b. a plurality of structured nucleic acid particles, wherein            each structured nucleic acid particle comprises a coupling            moiety, wherein the coupling moiety comprises a plurality of            oligonucleotides, wherein each oligonucleotide of the            plurality of oligonucleotides comprises a            surface-interacting moiety, wherein each structured nucleic            acid particle of the plurality of structured nucleic acid            particles is coupled to an address of the plurality of            addresses by a binding of the surface-interacting moiety of            the plurality of oligonucleotides to a surface-linked moiety            of the one or more complementary oligonucleotides, and            wherein a structured nucleic acid particle of the plurality            of structured nucleic acid particles comprises a display            moiety comprising a coupling site that is coupled to an            analyte.    -   300. The single-analyte array of clause 299, wherein the array        comprises an ordered array.    -   301. The single-analyte array of clause 300, wherein each        coupling surface is formed by a lithographic process.    -   302. The single-analyte array of clause 300 or 301, wherein each        address of the plurality of addresses is adjacent to one or more        interstitial regions, wherein each interstitial region of the        one or more interstitial regions does not comprise a coupling        surface.    -   303. The single-analyte array of clause 302, wherein an        interstitial region of the one or more interstitial regions        comprises a disrupting moiety, wherein the disrupting moiety is        configured to reduce the likelihood of a coupling of a molecule        to the interstitial region.    -   304. The single-analyte array of clause 302 or 303, wherein a        coupling surface comprises a raised surface or a depressed        surface relative to an interstitial region of the one or more        interstitial regions.    -   305. The single-analyte array of clause 299, wherein the array        comprises an unordered array.    -   306. The single-analyte array of clause 305, wherein the        unordered array further comprises a lipid bilayer adjacent to        the solid support.    -   307. The method of clause 306, wherein a surface-linked moiety        of the one or more surface-linked moieties is coupled to a lipid        molecule of the lipid bilayer.    -   308. The method of clause 307, wherein the lipid molecule        comprises a phospholipid or a cholesterol.    -   309. The single-analyte array of any one of clauses 299-308,        wherein a SNAP-occupied fraction of the plurality of addresses        comprises at least 0.5.    -   310. The single-analyte array of clause 309, wherein the        SNAP-occupied fraction of the plurality of addresses comprises        at least 0.9.    -   311. The single-analyte array of clause 309 or 310, wherein a        fraction of addresses of the plurality of addresses comprising        two or more SNAPs is no more than about 0.1.    -   312. The single-analyte array of clause 311, wherein the        fraction of addresses of the plurality of addresses comprising        two or more SNAPs is no more than about 0.01.    -   313. The single-analyte array of any one of clauses 309-312,        wherein a fraction of addresses with a detectable analyte is at        least 0.5.    -   314. The single-analyte array of clause 313, wherein the        fraction of addresses with a detectable analyte is at least 0.9.    -   315. A single-analyte array, comprising:        -   a. a solid support comprising a plurality of addresses,            wherein each address of the plurality of addresses is            resolvable from each other address at single-analyte            resolution, and wherein each address is separated from each            adjacent address by one or more interstitial regions; and        -   b. a plurality of analytes, wherein a single analyte of the            plurality of analytes is coupled to an address of the            plurality of addresses, wherein each address of the            plurality of addresses comprises no more than one single            analyte, wherein each single analyte is coupled to a            coupling surface of the address by a nucleic acid structure,            and wherein the nucleic acid structure occludes the single            analyte from contacting the coupling surface.

What is claimed is:
 1. A method of preparing a proteomic array,comprising: (a) coupling a plurality of sample polypeptides from aproteome to a plurality of nucleic acid nanostructures, wherein eachnucleic acid nanostructure is coupled to one and only one samplepolypeptide by a separating group, wherein the separating group providesan average separation distance of at least 10 nanometers between the oneand only one sample polypeptide and a nucleic acid nanostructure towhich the one and only sample polypeptide is coupled, and wherein theplurality of nucleic acid nanostructures is coupled to at least 25% ofall polypeptide species in the proteome; (b) after coupling theplurality of sample polypeptides to the plurality of nucleic acidnanostructures, coupling the plurality of nucleic acid nanostructures toan array comprising a plurality of sites; and (c) after coupling theplurality of nucleic acid nanostructures to the array, detectingpresence or absence of a nucleic acid nanostructure at each site of theplurality of sites.
 2. The method of claim 1, wherein detecting thepresence or absence of a nucleic acid nanostructure at each site of theplurality of sites comprises detecting the presence of a nucleic acidnanostructure for at least 65% of sites of the plurality of sites. 3.The method of claim 1, wherein detecting the presence or absence of anucleic acid nanostructure at each site of the plurality of sitescomprises detecting the absence of a nucleic acid nanostructure for nomore than 35% of sites of the plurality of sites.
 4. The method of claim1, wherein, after coupling the plurality of nucleic acid nanostructuresto the array, at least 40% of sites of the plurality of sites compriseone and only one sample polypeptide.
 5. The method of claim 1, whereindetecting presence or absence of a nucleic acid nanostructure at eachsite of the plurality of sites comprises detecting the presence orabsence of a first signal from a first detectable label coupled to anucleic acid nanostructure.
 6. The method of claim 1, furthercomprising: i) coupling affinity agents to sites of the plurality ofsites, and ii) detecting presence or absence of an affinity agent ateach site of the plurality of sites.
 7. The method of claim 6, whereindetecting the presence or absence of the affinity agent at each site ofthe plurality of sites comprises detecting the presence or absence of asignal from a detectable label coupled to the affinity agent.
 8. Themethod of claim 6, further comprising detecting coupling of affinityagents to at least 50% of the sample polypeptides on the proteomicarray.
 9. The method of claim 8, further comprising identifying at least50% of sample polypeptides of the plurality of sample polypeptides basedupon detected coupling of affinity agents to the at least 50% of thesample polypeptides.
 10. The method of claim 1, wherein coupling theplurality of sample polypeptides to the plurality of nucleic acidnanostructures comprises covalently coupling the plurality of samplepolypeptides to the plurality of nucleic acid nanostructures.
 11. Themethod of claim 10, wherein nucleic acid nanostructures of the pluralityof nucleic acid nanostructures comprises linking moieties, and whereincovalently coupling the plurality of sample polypeptides to theplurality of nucleic acid nanostructures comprises covalently couplingsample polypeptides to the linking moieties.
 12. The method of claim 1,further comprising obtaining the plurality of sample polypeptides froman organism.
 13. The method of claim 12, comprising obtaining theplurality of sample polypeptides from a tissue or a biological fluid ofthe organism.
 14. The method of claim 12, wherein the organism comprisesa mammal or a plant.
 15. The method of claim 1, further comprisingdetecting a terminal amino acid residue of a sample polypeptide on thearray.
 16. The method of claim 1, wherein coupling a nucleic acidnanostructure of the plurality of nucleic acid nanostructures to a siteof the plurality of sites comprises hybridizing a nucleic acid strand ofthe nucleic acid nanostructure to a complementary nucleic acid strand,wherein the complementary nucleic acid strand is coupled to the site.17. The method of claim 1, wherein a nucleic acid nanostructure of theplurality of nucleic acid nanostructures comprises a nucleic acidorigami.
 18. The method of claim 17, wherein the nucleic acidnanostructure of the plurality of nucleic acid nanostructures furthercomprises a pervious structure, wherein the pervious structure comprisesa plurality of pendant moieties.
 19. The method of claim 18, whereincoupling a nucleic acid nanostructure of the plurality of nucleic acidnanostructures to a site of the plurality of sites comprises coupling apendant moiety of the plurality of pendant moieties to a site of theplurality of sites.
 20. The method of claim 18, wherein coupling anucleic acid nanostructure of the plurality of nucleic acidnanostructures to a site of the plurality of sites comprises coupling asubset of pendant moieties of the plurality of pendant moieties to asite of the plurality of sites.
 21. The method of claim 18, wherein apendant moiety of the plurality of pendant moieties comprises asingle-stranded nucleic acid.
 22. The method of claim 18, wherein apendant moiety of the plurality of pendant moieties comprises a polymerchain.
 23. The method of claim 1, further comprising: i) fragmenting asample polypeptide into at least one peptide fragment, and ii) couplinga peptide fragment of the at least one peptide fragment to a nucleicacid nanostructure.
 24. The method of claim 1, wherein the at least 25%of species of the proteome comprise a complexity of at least about 1×10³native-length primary protein sequences.
 25. The method of claim 1,wherein the separating group comprises a polymer linker, a nucleic acidlinker, or a nanoparticle linker.
 26. The method of claim 25, whereinthe polymer linker comprises a polyethylene glycol (PEG) moiety.
 27. Themethod of claim 25, wherein the nucleic acid linker comprises adouble-stranded nucleic acid.
 28. The method of claim 1, wherein theseparating group provides an average separation distance of at least 15nanometers between the one and only one sample polypeptide and a surfaceof a solid support.
 29. A method of preparing a proteomic array,comprising: a) coupling a plurality of sample polypeptides from aproteome to a plurality of nucleic acid nanostructures, wherein eachnucleic acid nanostructure is coupled to one and only one samplepolypeptide, wherein the coupling occurs in the presence of a denaturingspecies, and wherein the plurality of nucleic acid nanostructures iscoupled to at least 25% of all polypeptide species in the proteome; b)after coupling the plurality of sample polypeptides from the proteome tothe plurality of nucleic acid nanostructures, coupling the plurality ofnucleic acid nanostructures to an array comprising a plurality of sites;and c) after coupling the plurality of nucleic acid nanostructures tothe array, detecting presence or absence of a nucleic acid nanostructureat each site of the plurality of sites.
 30. A method of preparing aproteomic array, comprising: a) coupling a plurality of samplepolypeptides from a proteome to a plurality of nucleic acidnanostructures, wherein each nucleic acid nanostructure is coupled toone and only one sample, and wherein the plurality of nucleic acidnanostructures is coupled to at least 25% of all polypeptide species inthe proteome; b) after coupling the plurality of sample polypeptidesfrom the proteome to the plurality of nucleic acid nanostructures,coupling the plurality of nucleic acid nanostructures to an arraycomprising a plurality of sites, wherein coupling a nucleic acidnanostructure of the plurality of nucleic acid nanostructures compriseshybridizing a nucleic acid strand of the nucleic acid nanostructure totwo or more complementary nucleic acid strands of a site of theplurality of sites, wherein the two or more complementary nucleic acidstrands are coupled to the site; and c) after coupling the plurality ofnucleic acid nanostructures to the array, detecting presence or absenceof a nucleic acid nanostructure at each site of the plurality of sites.